Dehydrogenase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to 3-hydroxypropionate dehydrogenase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/242,721, filed Apr. 1, 2014, now U.S. Pat. No. 9,163,220, which is acontinuation of U.S. patent application Ser. No. 13/825,515, now U.S.Pat. No. 8,728,788, which is a 35 U.S.C. §371 national application ofPCT/US2012/057134, filed Sep. 25, 2012, which claims priority to U.S.Provisional Application No. 61/541,363, filed Sep. 30, 2011. Thecontents of these applications are hereby incorporated by reference intheir entireties.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND

3-hydroxypropionic acid (3-HP) is a three carbon carboxylic acididentified by the U.S. Department of Energy as one of the top 12high-potential building block chemicals that can be made byfermentation. Alternative names for 3-HP, which is an isomer of lactic(2-hydroxypropionic) acid, include ethylene lactic acid and3-hydroxypropionate. 3-HP is an attractive renewable platform chemical,with 100% theoretical yield from glucose, multiple functional groupsthat allow it to participate in a variety of chemical reactions, and lowtoxicity. 3-HP can be used as a substrate to form several commoditychemicals, such as 1,3-propanediol, malonic acid, acrylamide, andacrylic acid. Acrylic acid is a large-volume chemical (>7 billionlbs/year) used to make acrylate esters and superabsorbent polymers, andis currently derived from catalytic oxidation of propylene. Fermentativeproduction of 3-HP would provide a sustainable alternative topetrochemicals as the feedstock for these commercially-significantchemicals, thus reducing energy consumption, dependence on foreign oilsupplies, and the production of greenhouse gases.

3-hydroxypropionate dehydrogenase (3-HPDH) is an enzyme that convertsmalonate semialdehyde to 3-HP (FIG. 1). Certain 3-HPDH enzymes utilizethe cofactor NADP(H) (EC 1.1.1.298). However, it may be desirable withsome engineered metabolic pathways for 3-HPDH to utilize the cofactorNAD(H) rather than NADP(H) (e.g., to improve redox balance).Accordingly, there is a need in the art to develop dehydrogenasevariants that have increased specificity for the cofactor NAD(H)compared to NADP(H). Described herein are dehydrogenase variants thatmeet this need.

SUMMARY

Described herein are 3-hydroxypropionate dehydrogenase variantscomprising a substitution at one or more (e.g., two, several) positionscorresponding to positions 9, 31, 32, 33, 34, 35 and 36 of SEQ ID NO: 2,wherein the variants have 3-hydroxypropionate dehydrogenase activity. Insome aspects, the variants comprise a deletion at a positioncorresponding to position 10 of SEQ ID NO: 2. In some aspects, thevariants have increased specificity for the cofactor NAD(H) compared toNADP(H).

Also described are isolated polynucleotides encoding the variants;nucleic acid constructs, vectors, and host cells comprising thepolynucleotides; methods of producing 3-hydroxypropionic acid (3-HP)using the host cells comprising the polynucleotides; and methods ofproducing the variants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a pathway for generating 3-HP.

FIG. 2 shows an alignment of native dehydrogenase sequences for E. coliydfG, I. orientalis YMR226c, and S. cerevisiae YMR226c (SEQ ID NOs: 2,4, and 6, respectively). Residues involved in cofactor binding areunderlined. Residues involved in catalysis are boldfaced.

FIG. 3 shows a partial sequence alignment for the N-terminal region ofvariant dehydrogenases mut1-mut25 (SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13,14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and33, respectively) compared to the native E. coli dehydrogenase (SEQ IDNO: 2); and for the N-terminal region of variant dehydrogenases mut26and mut27 (SEQ ID NOs: 80 and 81, respectively) compared to the nativeI. orientalis dehydrogenase (SEQ ID NO: 4).

FIG. 4 shows plasmid map for pTrc99A.

FIG. 5 shows plasmid map for pMcTs108.

FIG. 6 shows plasmid map for pMcTs116.

FIG. 7 shows plasmid map for p1045168.

FIG. 8 shows plasmid map for pMcTs77.

FIG. 9 shows plasmid map for p11AAT5WP.

FIG. 10 shows plasmid map for pMcTs78.

FIG. 11 shows plasmid map for pMcTs115.

FIG. 12 shows plasmid map for p11AA2GJP.

FIG. 13 shows plasmid map for pMcTs102.

DEFINITIONS

3-hydroxypropionate dehydrogenase: The term “3-hydroxypropionatedehydrogenase” (3-HPDH) means an enzyme that catalyzes theinterconversion of malonate semialdehyde to 3-hydroxypropionate (3-HP)in the presence of a NAD(H) or NADP(H) cofactor. Enzymes having 3-HPdehydrogenase activity are classified as EC 1.1.1.59 if they utilize anNAD(H) cofactor, and as EC 1.1.1.298 if they utilize an NADP(H)cofactor. Enzymes classified as EC 1.1.1.298 are alternatively referredto as malonate semialdehyde reductases. One skilled in the art willrecognize that 3-hydroxypropionate dehydrogenases may have specificityfor more than one substrate. For example, the E. coli3-hydroxypropionate dehydrogenase of SEQ ID NO: 2 may catalyze both theinterconversion of serine to 2-aminomalonate semialdehyde (i.e. a“serine dehydrogenase”) and the interconversion of 3-HP to malonatesemialdehyde (i.e., a 3-HPDH).

3-hydroxypropionate dehydrogenase activity can be determined accordingto malonate semi-aldehyde reductase assay described in the Examples. Inone aspect, the variants of the present invention have at least 20%,e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% of the3-hydroxypropionate dehydrogenase of SEQ ID NO: 2, 4, or 6.

Active 3-HP pathway: As used herein, a host cell having an “active 3-HPpathway” produces active enzymes necessary to catalyze each reaction ina metabolic pathway from a fermentable sugar to 3-HP, and therefore iscapable of producing 3-HP in measurable yields when cultivated underfermentation conditions in the presence of at least one fermentablesugar. A host cell having an active 3-HP pathway comprises one or more3-HP pathway genes. A “3-HP pathway gene” as used herein refers to agene that encodes an enzyme involved in an active 3-HP pathway. Oneexample of an active 3-HP pathway and corresponding enzymes involved inthe active 3-HP pathway is shown in FIG. 1.

The active enzymes necessary to catalyze each reaction in active 3-HPpathway may result from activities of endogenous gene expression,activities of heterologous gene expression, or from a combination ofactivities of endogenous and heterologous gene expression.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotidesequence, which specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a sequence ofgenomic DNA, cDNA, a synthetic polynucleotide, and/or a recombinantpolynucleotide.

Control sequence: The term “control sequence” means a nucleic acidsequence necessary for polypeptide expression. Control sequences may benative or foreign to the polynucleotide encoding the polypeptide, andnative or foreign to each other. Such control sequences include, but arenot limited to, a leader sequence, polyadenylation sequence, propeptidesequence, promoter sequence, signal peptide sequence, and transcriptionterminator sequence. The control sequences may be provided with linkersfor the purpose of introducing specific restriction sites facilitatingligation of the control sequences with the coding region of thepolynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion. Expression can bemeasured—for example, to detect increased expression—by techniques knownin the art, such as measuring levels of mRNA and/or translatedpolypeptide.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences, wherein thecontrol sequences provide for expression of the polynucleotide encodingthe polypeptide. At a minimum, the expression vector comprises apromoter sequence, and transcriptional and translational stop signalsequences.

Fermentable medium: The term “fermentable medium” refers to a mediumcomprising one or more (e.g., two, several) sugars, such as glucose,fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose,galactose, and/or soluble oligosaccharides, wherein the medium iscapable, in part, of being converted (fermented) into 3-HP by a hostcell having an active 3-HP pathway. In some instances, the fermentationmedium is derived from a natural source, such as sugar cane, starch, orcellulose, and may be the result of pretreating the source by enzymatichydrolysis (saccharification).

Fragment: The term “fragment” means a polypeptide having one or more(e.g., two, several) amino acids deleted from the amino and/or carboxylterminus of a referenced polypeptide sequence. In one aspect, thefragment has 3-HPDH activity. In another aspect, the number of aminoacid residues in the fragment is at least 75%, e.g., at least 80%, 85%,90%, or 95% of any 3-HPDH herein, e.g., at least 75%, e.g., at least80%, 85%, 90%, or 95% of the number of amino acid residues in SEQ IDNOs: 2, 4, or 6.

Heterologous polynucleotide: The term “heterologous polynucleotide” isdefined herein as a polynucleotide that is not native to the host cell;a native polynucleotide in which one or more (e.g., two, several)structural modifications have been made to the coding region; a nativepolynucleotide whose expression is quantitatively altered as a result ofmanipulation of the DNA by recombinant DNA techniques, e.g., a different(foreign) promoter linked to the polynucleotide; or a nativepolynucleotide whose expression is quantitatively altered by theintroduction of one or more extra copies of the polynucleotide into thehost cell.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotidedescribed herein (e.g., a polynucleotide encoding a 3-HPDH). The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication.

Increased specificity: The term “increased specificity for NAD(H)compared to NADP(H)” means the referenced polypeptide has greater 3-HPDHactivity in the presence of NAD(H) compared to NADP(H) in otherwiseidentical conditions. In some aspects, the referenced variant has morethan 2-fold, e.g., more than 5-fold, 10-fold, 20-fold, 50-fold,100-fold, 200-fold, 500-fold, or 1000-fold specificity for NAD(H)compared to NADP(H).

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means apolynucleotide that comprises one or more (e.g., two, several) controlsequences. The polynucleotide may be single-stranded or double-stranded,and may be isolated from a naturally occurring gene, modified to containsegments of nucleic acids in a manner that would not otherwise exist innature, or synthetic.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Parent or parent 3-HPDH: The term “parent” or “parent 3-HPDH” means a3-HPDH to which an alteration is made to produce the enzyme variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix.

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix.

Stringency conditions: Stringency conditions are used herein to providehybridization conditions when comparing two DNA sequences.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., two, several) nucleotides deleted from the 5′ and/or 3′ endof the referenced nucleotide sequence. In one aspect, the subsequenceencodes a fragment having 3-HPDH activity. In another aspect, the numberof nucleotides residues in the subsequence is at least 75%, e.g., atleast 80%, 85%, 90%, or 95% of the number of nucleotide residues in anysequence encoding a 3-HPDH described herein, e.g., at least 75%, e.g.,at least 80%, 85%, 90%, or 95% of the number of nucleotide residues inSEQ ID NOs: 1, 3, or 5.

Variant: The term “variant” means a 3-HPDH comprising an alteration,i.e., a substitution, insertion, and/or deletion, at one or more (e.g.,two, several) positions relative to a parent 3-HPDH. A substitutionmeans replacement of the amino acid occupying a position with adifferent amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Wild-type: The term “wild-type” 3-HPDH or “native” 3-HPDH means a 3-HPDHexpressed by a naturally occurring microorganism, such as a bacterium,yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes described herein, SEQ ID NO: 2 is used to determine aminoacid numbering in other 3-HPDH enzymes. The amino acid sequence ofanother 3-HPDH is aligned with SEQ ID NO: 2, and based on the alignment,the amino acid position number corresponding to any amino acid residuein the polypeptide disclosed in SEQ ID NO: 2 is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in another 3-HPDHcan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the other enzyme sequence has diverged from the SEQ ID NO: 2 suchthat traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants described herein, the nomenclature describedbelow is adapted for ease of reference. The accepted IUPAC single letteror three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Alternative substitutions at the same positionare separated by a slant. For example, the substitution of threonine atposition 226 with alanine or valine is designated as “Thr226Ala/Val” or“T226A/V”, representing a T226A or T226V substitution. Multiplemutations are separated by addition marks (“+”), e.g.,“Gly205Arg+Ser411Phe/Tyr” or “G205R+S411F/Y”, representing substitutionsat positions 205 and 411 of glycine (G) with arginine (R) and serine (S)with phenylalanine (F) or tyrosine (Y), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Reference to “about” a value or parameter herein includes aspects thatare directed to that value or parameter per se. For example, descriptionreferring to “about X” includes the aspect “X”. When used in combinationwith measured values, “about” includes a range that encompasses at leastthe uncertainty associated with the method of measuring the particularvalue, and can include a range of plus or minus two standard deviationsaround the stated value.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that the aspects described herein include“consisting” and/or “consisting essentially of” aspects.

Unless defined otherwise or clearly indicated by context, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art.

DETAILED DESCRIPTION

Described herein, inter alia, are polypeptides having 3-HPDH activity.In some aspects, the polypeptides are variants comprising a substitutionat one or more (e.g., two, several) positions corresponding to positions9, 31, 32, 33, 34, 35 and 36 of SEQ ID NO: 2. In some aspects thevariants further comprise a deletion at a position corresponding toposition 10 of SEQ ID NO: 2. In some aspects, the polypeptides havingincreased specificity for NAD(H) compared to NADP(H).

Polypeptides Having 3-HPDH Activity

In one aspect is a polypeptide having 3-HPDH activity, wherein thepolypeptide is:

a) a polypeptide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to SEQ ID NO: 2, 4, or 6;

b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, e.g., medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with the full-length complementary strand of SEQID NO: 1, 3, or 5; or

c) a polypeptide encoded by a polynucleotide having at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, sequence identity to SEQ ID NO: 1, 3, or 5;

wherein the polypeptide has increased specificity for NAD(H) compared toNADP(H) (e.g., greater than 2-fold, 5-fold, 10-fold, 20-fold, 50-fold,100-fold, 200-fold, 500-fold, or 1000-fold specificity for NAD(H)compared to NADP(H)).

In one aspect, the polypeptide a) has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 2; b) is encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with thefull-length complementary strand of SEQ ID NO: 1; and/or c) is encodedby a polynucleotide having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1.

In some of these aspects related to SEQ ID NO: 2, at least one ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differs from SEQ ID NO: 2. In one embodiment, at least two of positions9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ fromSEQ ID NO: 2. In another embodiment, at least three of positions 9, 31,32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ IDNO: 2. In another embodiment, at least four of positions 9, 31, 32, 33,34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 2. Inanother embodiment, at least five of positions 9, 31, 32, 33, 34, 35 and36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 2. In anotherembodiment, at least six of positions 9, 31, 32, 33, 34, 35 and 36corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 2. In anotherembodiment, all of positions 9, 31, 32, 33, 34, 35 and 36 correspondingto SEQ ID NO: 2 differ from SEQ ID NO: 2.

In another aspect, the polypeptide a) has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to SEQ ID NO: 4; b) is encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with thefull-length complementary strand of SEQ ID NO: 3; and/or c) is encodedby a polynucleotide having at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 3.

In some of these aspects related to SEQ ID NO: 4, at least one ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differs from SEQ ID NO: 4. In one embodiment, at least two of positions9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ fromSEQ ID NO: 4. In another embodiment, at least three of positions 9, 31,32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ IDNO: 4. In another embodiment, at least four of positions 9, 31, 32, 33,34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 4. Inanother embodiment, at least five of positions 9, 31, 32, 33, 34, 35 and36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 4. In anotherembodiment, at least six of positions 9, 31, 32, 33, 34, 35 and 36corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 4. In anotherembodiment, all of positions 9, 31, 32, 33, 34, 35 and 36 correspondingto SEQ ID NO: 2 differ from SEQ ID NO: 4.

In another aspect, the polypeptide a) at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 6; b) is encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with thefull-length complementary strand of SEQ ID NO: 5; and/or is encoded by apolynucleotide having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 5.

In some of these aspects related to SEQ ID NO: 6, at least one ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differs from SEQ ID NO: 6. In one embodiment, at least two of positions9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ fromSEQ ID NO: 6. In another embodiment, at least three of positions 9, 31,32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ IDNO: 6. In another embodiment, at least four of positions 9, 31, 32, 33,34, 35 and 36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 6. Inanother embodiment, at least five of positions 9, 31, 32, 33, 34, 35 and36 corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 6. In anotherembodiment, at least six of positions 9, 31, 32, 33, 34, 35 and 36corresponding to SEQ ID NO: 2 differ from SEQ ID NO: 6. In anotherembodiment, all of positions 9, 31, 32, 33, 34, 35 and 36 correspondingto SEQ ID NO: 2 differ from SEQ ID NO: 6.

In one aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, orVal at a position corresponding to position 9 of SEQ ID NO: 2. In someembodiments, the amino acid corresponding to position 9 is Gly. In someembodiments, the polypeptide comprises a deletion at a positioncorresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 31 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 31 is Aspor Glu. In some embodiments, the polypeptide comprises a deletion at aposition corresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 32 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 32 is Leu.In some embodiments, the polypeptide comprises a deletion at a positioncorresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 33 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 33 is Seror Asn. In some embodiments, the polypeptide comprises a deletion at aposition corresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 34 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 34 is Alaor Pro. In some embodiments, the polypeptide comprises a deletion at aposition corresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 35 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 35 is Alaor Asp. In some embodiments, the polypeptide comprises a deletion at aposition corresponding to position 10.

In another aspect, the polypeptide may comprise an Ala, Arg, Asn, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val at a position corresponding to position 36 of SEQ ID NO: 2.In some embodiments, the amino acid corresponding to position 36 is Ala.In some embodiments, the polypeptide comprises a deletion at a positioncorresponding to position 10.

In one aspect of the polypeptide, the position corresponding to 9 is Glyand 31 is Asp or Glu; 9 is Gly and 32 is Leu; 9 is Gly and 33 is Ser orAsn; 9 is Gly and 34 is Ala or Pro; 9 is Gly and 35 is Ala or Asp; 9 isGly and 36 is Ala; 31 is Asp or Glu and 32 is Leu; 31 is Asp or Glu and33 is Ser or Asn; 31 is Asp or Glu and 34 is Ala or Pro; 31 is Asp orGlu and 35 is Ala or Asp; 31 is Asp or Glu and 36 is Ala; 32 is Leu and33 is Ser or Asn; 32 is Leu and 34 is Ala or Pro; 32 is Leu and 35 isAla or Asp; 32 is Leu and 36 is Ala; 33 is Ser or Asn and 34 is Ala orPro; 33 is Ser or Asn and 35 is Ala or Asp; 33 is Ser or Asn and 36 isAla; 34 is Ala or Pro and 35 is Ala or Asp; 34 is Ala or Pro and 36 isAla; or 35 is Ala or Asp and 36 is Ala. In some embodiments, thepolypeptide comprises a deletion at a position corresponding to position10.

In another aspect of the polypeptide, the position corresponding to 9 isGly, 31 is Asp or Glu, and 32 is Leu; 9 is Gly, 31 is Asp or Glu, and 33is Ser or Asn; 9 is Gly, 31 is Asp or Glu, and 34 is Ala or Pro; 9 isGly, 31 is Asp or Glu, and 35 is Ala or Asp; 9 is Gly, 31 is Asp or Glu,and 36 is Ala; 9 is Gly, 32 is Leu, and 33 is Ser or Asn; 9 is Gly, 32is Leu, and 34 is Ala or Pro; 9 is Gly, 32 is Leu, and 35 is Ala or Asp;9 is Gly, 32 is Leu, and 36 is Ala; 9 is Gly, 33 is Ser or Asn, and 34is Ala or Pro; 9 is Gly, 33 is Ser or Asn, and 35 is Ala or Asp; 9 isGly, 33 is Ser or Asn, and 36 is Ala; 9 is Gly, 34 is Ala or Pro, and 35is Ala or Asp; 9 is Gly, 34 is Ala or Pro, and 36 is Ala; 9 is Gly, 35is Ala or Asp, and 36 is Ala; 31 is Asp or Glu, 32 is Leu, and 33 is Seror Asn; 31 is Asp or Glu, 32 is Leu, and 34 is Ala or Pro; 31 is Asp orGlu, 32 is Leu, and 35 is Ala or Asp; 31 is Asp or Glu, 32 is Leu, and36 is Ala; 31 is Asp or Glu, 33 is Ser or Asn, and 34 is Ala or Pro; 31is Asp or Glu, 33 is Ser or Asn, and 35 is Ala or Asp; 31 is Asp or Glu,33 is Ser or Asn, and 36 is Ala; 31 is Asp or Glu, 34 is Ala or Pro, and35 is Ala or Asp; 31 is Asp or Glu, 34 is Ala or Pro, and 36 is Ala; 31is Asp or Glu, 35 is Ala or Asp, and 36 is Ala; 32 is Leu, 33 is Ser orAsn, and 34 is Ala or Pro; 32 is Leu, 33 is Ser or Asn, and 35 is Ala orAsp; 32 is Leu, 33 is Ser or Asn, and 36 is Ala; 32 is Leu, 34 is Ala orPro, and 35 is Ala or Asp; 32 is Leu, 34 is Ala or Pro, and 36 is Ala;32 is Leu, 35 is Ala or Asp, and 36 is Ala; 33 is Ser or Asn, 34 is Alaor Pro, and 35 is Ala or Asp; 33 is Ser or Asn, 34 is Ala or Pro, and 36is Ala; 33 is Ser or Asn, 35 is Ala or Asp, and 36 is Ala; or 34 is Alaor Pro, 35 is Ala or Asp, and 36 is Ala. In some embodiments, thepolypeptide comprises a deletion at a position corresponding to position10.

In another aspect of the polypeptide, the position corresponding to 9 isGly, 31 is Asp or Glu, 32 is Leu, and 33 is Ser or Asn; 9 is Gly, 31 isAsp or Glu, 32 is Leu, and 34 is Ala or Pro; 9 is Gly, 31 is Asp or Glu,32 is Leu, and 35 is Ala or Asp; 9 is Gly, 31 is Asp or Glu, 32 is Leu,and 36 is Ala; 9 is Gly, 31 is Asp or Glu, 33 is Ser or Asn, and 34 isAla or Pro; 9 is Gly, 31 is Asp or Glu, 33 is Ser or Asn, and 35 is Alaor Asp; 9 is Gly, 31 is Asp or Glu, 33 is Ser or Asn, and 36 is Ala; 9is Gly, 31 is Asp or Glu, 34 is Ala or Pro, and 35 is Ala or Asp; 9 isGly, 31 is Asp or Glu, 34 is Ala or Pro, and 36 is Ala; 9 is Gly, 31 isAsp or Glu, 35 is Ala or Asp, and 36 is Ala; 9 is Gly, 32 is Leu, 33 isSer or Asn, and 34 is Ala or Pro; 9 is Gly, 32 is Leu, 33 is Ser or Asn,and 35 is Ala or Asp; 9 is Gly, 32 is Leu, 33 is Ser or Asn, and 36 isAla; 9 is Gly, 32 is Leu, 34 is Ala or Pro, and 35 is Ala or Asp; 9 isGly, 32 is Leu, 34 is Ala or Pro, and 36 is Ala; 9 is Gly, 32 is Leu, 35is Ala or Asp, and 36 is Ala; 9 is Gly, 33 is Ser or Asn, 34 is Ala orPro, and 35 is Ala or Asp; 9 is Gly, 33 is Ser or Asn, 34 is Ala or Pro,and 36 is Ala; 9 is Gly, 33 is Ser or Asn, 35 is Ala or Asp, and 36 isAla; 9 is Gly, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; 31 isAsp or Glu, 32 is Leu, 33 is Ser or Asn, and 34 is Ala or Pro; 31 is Aspor Glu, 32 is Leu, 33 is Ser or Asn, and 35 is Ala or Asp; 31 is Asp orGlu, 32 is Leu, 33 is Ser or Asn, and 36 is Ala; 31 is Asp or Glu, 32 isLeu, 34 is Ala or Pro, and 35 is Ala or Asp; 31 is Asp or Glu, 32 isLeu, 34 is Ala or Pro, and 36 is Ala; 31 is Asp or Glu, 32 is Leu, 35 isAla or Asp, and 36 is Ala; 31 is Asp or Glu, 33 is Ser or Asn, 34 is Alaor Pro, and 35 is Ala or Asp; 31 is Asp or Glu, 33 is Ser or Asn, 34 isAla or Pro, and 36 is Ala; 31 is Asp or Glu, 33 is Ser or Asn, 35 is Alaor Asp, and 36 is Ala; 31 is Asp or Glu, 34 is Ala or Pro, 35 is Ala orAsp, and 36 is Ala; 32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro, and35 is Ala or Asp; 32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro, and 36is Ala; 32 is Leu, 33 is Ser or Asn, 35 is Ala or Asp, and 36 is Ala; 32is Leu, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; or 33 is Seror Asn, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala. In someembodiments, the polypeptide comprises a deletion at a positioncorresponding to position 10.

In another aspect of the polypeptide, the position corresponding to 9 isGly, 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, and 34 is Ala orPro; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, and 35 isAla or Asp; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, and36 is Ala; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 34 is Ala or Pro, and35 is Ala or Asp; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 34 is Ala orPro, and 36 is Ala; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 35 is Ala orAsp, and 36 is Ala; 9 is Gly, 31 is Asp or Glu, 33 is Ser or Asn, 34 isAla or Pro, and 35 is Ala or Asp; 9 is Gly, 31 is Asp or Glu, 33 is Seror Asn, 34 is Ala or Pro, and 36 is Ala; 9 is Gly, 31 is Asp or Glu, 33is Ser or Asn, 35 is Ala or Asp, and 36 is Ala; 9 is Gly, 31 is Asp orGlu, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; 9 is Gly, 32 isLeu, 33 is Ser or Asn, 34 is Ala or Pro, and 35 is Ala or Asp; 9 is Gly,32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro, and 36 is Ala; 9 is Gly,32 is Leu, 33 is Ser or Asn, 35 is Ala or Asp, and 36 is Ala; 9 is Gly,32 is Leu, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; 9 is Gly,33 is Ser or Asn, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; 31is Asp or Glu, 32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro, and 35 isAla or Asp; 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, 34 is Ala orPro, and 36 is Ala; 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, 35 isAla or Asp, and 36 is Ala; 31 is Asp or Glu, 32 is Leu, 34 is Ala orPro, 35 is Ala or Asp, and 36 is Ala; 31 is Asp or Glu, 33 is Ser orAsn, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; or 32 is Leu, 33is Ser or Asn, 34 is Ala or Pro, 35 is Ala or Asp, and 36 is Ala. Insome embodiments, the polypeptide comprises a deletion at a positioncorresponding to position 10.

In another aspect of the polypeptide, the position corresponding to 9 isGly, 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro,and 35 is Ala or Asp; 9 is Gly, 31 is Asp or Glu, 32 is Leu, 33 is Seror Asn, 34 is Ala or Pro, and 36 is Ala; 9 is Gly, 31 is Asp or Glu, 32is Leu, 33 is Ser or Asn, 35 is Ala or Asp, and 36 is Ala; 9 is Gly, 31is Asp or Glu, 32 is Leu, 34 is Ala or Pro, 35 is Ala or Asp, and 36 isAla; 9 is Gly, 31 is Asp or Glu, 33 is Ser or Asn, 34 is Ala or Pro, 35is Ala or Asp, and 36 is Ala; 9 is Gly, 32 is Leu, 33 is Ser or Asn, 34is Ala or Pro, 35 is Ala or Asp, and 36 is Ala; or 31 is Asp or Glu, 32is Leu, 33 is Ser or Asn, 34 is Ala or Pro, 35 is Ala or Asp, and 36 isAla. In some embodiments, the polypeptide comprises a deletion at aposition corresponding to position 10.

In another aspect of the polypeptide, the position corresponding to 9 isGly, 31 is Asp or Glu, 32 is Leu, 33 is Ser or Asn, 34 is Ala or Pro, 35is Ala or Asp and 36 is Ala of SEQ ID NO: 2. In some embodiments, thepolypeptide comprises a deletion at a position corresponding to position10.

In any of these aspects, the polypeptide may have increased specificityfor NAD(H) compared to NADP(H). In some embodiments, the polypeptide hasmore than 2-fold, e.g., more than 5-fold, 10-fold, 20-fold, 50-fold,100-fold, 200-fold, 500-fold, or 1000-fold specificity for NAD(H)compared to NADP(H).

Variants

In some aspects, the polypeptides are described as 3-HPDH variants of aparent 3-HPDH, comprising substitutions at one or more (e.g., two,several) positions corresponding to any of positions 9, 31, 32, 33, 34,35 and 36 of SEQ ID NO: 2, wherein the variants have 3-HPDH activity.For example, the variants may comprise one or more (e.g., two, several)of the substitutions T/S9G, G/A31D/E, R32L, R33S/N, L/K/Q34A/P, E35D/A,and K/R36A. In some aspects, the variants further comprise a deletion ata position corresponding to position 10. In some aspects, the variantsare isolated.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, to sequence identity to a parent 3-HPDH.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 4.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 6.

In one aspect, the number of alterations (substitutions, deletions,and/or insertions) in the variants of the described herein is 1-20,e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 9 of SEQ ID NO: 2. For example, theamino acid corresponding to position 9 may be substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In one aspect, the amino acid corresponding toposition 9 is Gly. In another aspect, the variant comprises or consistsof the substitution T/S9G, such as the substitution T9G of a parentcomprising SEQ ID NO: 2, or S9G of a parent comprising SEQ ID NO: 4 or6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 31 of SEQ ID NO: 2. For example, theamino acid corresponding to position 31 may be substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 31 is Asp or Glu. In another aspect, the variant comprisesor consists of the substitution G/A31 D/E, such as the substitution G31D/E of a parent comprising SEQ ID NO: 2, or A31 D/E of a parentcomprising SEQ ID NO: 4 or 6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 32 of SEQ ID NO: 2. For example, theamino acid corresponding to position 32 may be substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 32 is Leu. In another aspect, the variant comprises orconsists of the substitution R32L, such as the substitution R32L of aparent comprising SEQ ID NO: 2, 4, or 6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 33 of SEQ ID NO: 2. For example, theamino acid corresponding to position 33 may be substituted with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 33 is Ser or Asn. In another aspect, the variant comprisesor consists of the substitution R33S/N, such as the substitution R33S/Nof a parent comprising SEQ ID NO: 2, 4, or 6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 34 of SEQ ID NO: 2. For example, theamino acid corresponding to position 34 may be substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 34 is Ala or Pro. In another aspect, the variant comprisesor consists of the substitution L/K/Q34A/P, such as the substitutionQ34A/P of a parent comprising SEQ ID NO: 2, KQ34A/P of a parentcomprising SEQ ID NO: 2, or L34A/P of a parent comprising SEQ ID NO: 6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 35 of SEQ ID NO: 2. For example, theamino acid corresponding to position 35 may be substituted with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 35 is Ala or Asp. In another aspect, the variant comprisesor consists of the substitution E35D/A, such as the substitution E35D/Aof a parent comprising SEQ ID NO: 2, 4, or 6.

In one aspect, the variant comprises or consists of a substitution at aposition corresponding to position 36 of SEQ ID NO: 2. For example, theamino acid corresponding to position 36 may be substituted with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In one aspect, the amino acid correspondingto position 36 is Ala. In another aspect, the variant comprises orconsists of the substitution K/R36A, such as the substitution R36A of aparent comprising SEQ ID NO: 2, or K36A of a parent comprising SEQ IDNO: 4 or 6.

In another aspect, the variant comprises or consists of substitutions atany two positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2. For example, the variant may comprise or consist oftwo substitutions corresponding to positions 9 and 31; 9 and 32; 9 and33; 9 and 34; 9 and 35; 9 and 36; 31 and 32; 31 and 33; 31 and 34; 31and 35; 31 and 36; 32 and 33; 32 and 34; 32 and 35; 32 and 36; 33 and34; 33 and 35; 33 and 36; 34 and 35; 34 and 36; or 35 and 36, such asthose described above. In one aspect, the variant comprises or consistsof the substitutions T/S9G and G/A31 D/E; T/S9G and R32L; T/S9G andR33S/N; T/S9G and L/K/Q34A/P; T/S9G and E35D/A; T/S9G and K/R36A;G/A31D/E and R32L; G/A31D/E and R33S/N; G/A31D/E and L/K/Q34A/P;G/A31D/E and E35D/A; G/A31D/E and K/R36A; R32L and R33S/N; R32L andL/K/Q34A/P; R32L and E35D/A; R32L and K/R36A; R33S/N and L/K/Q34A/P;R33S/N and E35D/A; R33S/N and K/R36A; L/K/Q34A/P and E35D/A; L/K/Q34A/Pand K/R36A; or E35D/A and K/R36A. In any of these aspects, the variantmay further comprise a deletion at a position corresponding to position10 of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of substitutions atany three positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2. For example, the variant may comprise or consist ofthree substitutions corresponding to positions 9, 31, and 32; 9, 31, and33; 9, 31, and 34; 9, 31, and 35; 9, 31, and 36; 9, 32, and 33; 9, 32,and 34; 9, 32, and 35; 9, 32, and 36; 9, 33, and 34; 9, 33, and 35; 9,33, and 36; 9, 34, and 35; 9, 34, and 36; 9, 35, and 36; 31, 32, and 33;31, 32, and 34; 31, 32, and 35; 31, 32, and 36; 31, 33, and 34; 31, 33,and 35; 31, 33, and 36; 31, 34, and 35; 31, 34, and 36; 31, 35, and 36;32, 33, and 34; 32, 33, and 35; 32, 33, and 36; 32, 34, and 35; 32, 34,and 36; 32, 35, and 36; 33, 34, and 35; 33, 34, and 36; 33, 35, and 36;or 34, 35, and 36, such as those described above. In one aspect, thevariant comprises or consists of the substitutions T/S9G, G/A31D/E, andR32L; T/S9G, G/A31D/E, and R33S/N; T/S9G, G/A31D/E, and L/K/Q34A/P;T/S9G, G/A31D/E, and E35D/A; T/S9G, G/A31D/E, and K/R36A; T/S9G, R32L,and R33S/N; T/S9G, R32L, and L/K/Q34A/P; T/S9G, R32L, and E35D/A; T/S9G,R32L, and K/R36A; T/S9G, R33S/N, and L/K/Q34A/P; T/S9G, R33S/N, andE35D/A; T/S9G, R33S/N, and K/R36A; T/S9G, L/K/Q34A/P, and E35D/A; T/S9G,L/K/Q34A/P, and K/R36A; T/S9G, E35D/A, and K/R36A; G/A31 D/E, R32L, andR33S/N; G/A31D/E, R32L, and L/K/Q34A/P; G/A31D/E, R32L, and E35D/A;G/A31D/E, R32L, and K/R36A; G/A31D/E, R33S/N, and L/K/Q34A/P; G/A31D/E,R33S/N, and E35D/A; G/A31D/E, R33S/N, and K/R36A; G/A31D/E, L/K/Q34A/P,and E35D/A; G/A31D/E, L/K/Q34A/P, and K/R36A; G/A31D/E, E35D/A, andK/R36A; R32L, R33S/N, and L/K/Q34A/P; R32L, R33S/N, and E35D/A; R32L,R33S/N, and K/R36A; R32L, L/K/Q34A/P, and E35D/A; R32L, L/K/Q34A/P, andK/R36A; R32L, E35D/A, and K/R36A; R33S/N, L/K/Q34A/P, and E35D/A;R33S/N, L/K/Q34A/P, and K/R36A; R33S/N, E35D/A, and K/R36A; orL/K/Q34A/P, E35D/A, and K/R36A. In any of these aspects, the variant mayfurther comprise a deletion at a position corresponding to position 10of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of substitutions atany four positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2. For example, the variant may comprise or consist offour substitutions corresponding to positions 9, 31, 32, and 33; 9, 31,32, and 34; 9, 31, 32, and 35; 9, 31, 32, and 36; 9, 31, 33, and 34; 9,31, 33, and 35; 9, 31, 33, and 36; 9, 31, 34, and 35; 9, 31, 34, and 36;9, 31, 35, and 36; 9, 32, 33, and 34; 9, 32, 33, and 35; 9, 32, 33, and36; 9, 32, 34, and 35; 9, 32, 34, and 36; 9, 32, 35, and 36; 9, 33, 34,and 35; 9, 33, 34, and 36; 9, 33, 35, and 36; 9, 34, 35, and 36; 31, 32,33, and 34; 31, 32, 33, and 35; 31, 32, 33, and 36; 31, 32, 34, and 35;31, 32, 34, and 36; 31, 32, 35, and 36; 31, 33, 34, and 35; 31, 33, 34,and 36; 31, 33, 35, and 36; 31, 34, 35, and 36; 32, 33, 34, and 35; 32,33, 34, and 36; 32, 33, 35, and 36; 32, 34, 35, and 36; or 33, 34, 35,and 36, such as those described above. In one aspect, the variantcomprises or consists of the substitutions T/S9G, G/A31D/E, R32L, andR33S/N; T/S9G, G/A31D/E, R32L, and L/K/Q34A/P; T/S9G, G/A31D/E, R32L,and E35D/A; T/S9G, G/A31D/E, R32L, and K/R36A; T/S9G, G/A31D/E, R33S/N,and L/K/Q34A/P; T/S9G, G/A31D/E, R33S/N, and E35D/A; T/S9G, G/A31D/E,R33S/N, and K/R36A; T/S9G, G/A31D/E, L/K/Q34A/P, and E35D/A; T/S9G,G/A31D/E, L/K/Q34A/P, and K/R36A; T/S9G, G/A31D/E, E35D/A, and K/R36A;T/S9G, R32L, R33S/N, and L/K/Q34A/P; T/S9G, R32L, R33S/N, and E35D/A;T/S9G, R32L, R33S/N, and K/R36A; T/S9G, R32L, L/K/Q34A/P, and E35D/A;T/S9G, R32L, L/K/Q34A/P, and K/R36A; T/S9G, R32L, E35D/A, and K/R36A;T/S9G, R33S/N, L/K/Q34A/P, and E35D/A; T/S9G, R33S/N, L/K/Q34A/P, andK/R36A; T/S9G, R33S/N, E35D/A, and K/R36A; T/S9G, L/K/Q34A/P, E35D/A,and K/R36A; G/A31D/E, R32L, R33S/N, and L/K/Q34A/P; G/A31D/E, R32L,R33S/N, and E35D/A; G/A31D/E, R32L, R33S/N, and K/R36A; G/A31D/E, R32L,L/K/Q34A/P, and E35D/A; G/A31D/E, R32L, L/K/Q34A/P, and K/R36A;G/A31D/E, R32L, E35D/A, and K/R36A; G/A31D/E, R33S/N, L/K/Q34A/P, andE35D/A; G/A31 D/E, R33S/N, L/K/Q34A/P, and K/R36A; G/A31 D/E, R33S/N,E35D/A, and K/R36A; G/A31D/E, L/K/Q34A/P, E35D/A, and K/R36A; R32L,R33S/N, L/K/Q34A/P, and E35D/A; R32L, R33S/N, L/K/Q34A/P, and K/R36A;R32L, R33S/N, E35D/A, and K/R36A; R32L, L/K/Q34A/P, E35D/A, and K/R36A;or R33S/N, L/K/Q34A/P, E35D/A, and K/R36A. In any of these aspects, thevariant may further comprise a deletion at a position corresponding toposition 10 of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of substitutions atany five positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2. For example, the variant may comprise or consist offive substitutions corresponding to positions 9, 31, 32, 33, and 34; 9,31, 32, 33, and 35; 9, 31, 32, 33, and 36; 9, 31, 32, 34, and 35; 9, 31,32, 34, and 36; 9, 31, 32, 35, and 36; 9, 31, 33, 34, and 35; 9, 31, 33,34, and 36; 9, 31, 33, 35, and 36; 9, 31, 34, 35, and 36; 9, 32, 33, 34,and 35; 9, 32, 33, 34, and 36; 9, 32, 33, 35, and 36; 9, 32, 34, 35, and36; 9, 33, 34, 35, and 36; 31, 32, 33, 34, and 35; 31, 32, 33, 34, and36; 31, 32, 33, 35, and 36; 31, 32, 34, 35, and 36; 31, 33, 34, 35, and36; or 32, 33, 34, 35, and 36, such as those described above. In oneaspect, the variant comprises or consists of the substitutions T/S9G,G/A31D/E, R32L, R33S/N, and L/K/Q34A/P; T/S9G, G/A31D/E, R32L, R33S/N,and E35D/A; T/S9G, G/A31D/E, R32L, R33S/N, and K/R36A; T/S9G, G/A31D/E,R32L, L/K/Q34A/P, and E35D/A; T/S9G, G/A31 D/E, R32L, L/K/Q34A/P, andK/R36A; T/S9G, G/A31 D/E, R32L, E35D/A, and K/R36A; T/S9G, G/A31D/E,R33S/N, L/K/Q34A/P, and E35D/A; T/S9G, G/A31D/E, R33S/N, L/K/Q34A/P, andK/R36A; T/S9G, G/A31D/E, R33S/N, E35D/A, and K/R36A; T/S9G, G/A31D/E,L/K/Q34A/P, E35D/A, and K/R36A; T/S9G, R32L, R33S/N, L/K/Q34A/P, andE35D/A; T/S9G, R32L, R33S/N, L/K/Q34A/P, and K/R36A; T/S9G, R32L,R33S/N, E35D/A, and K/R36A; T/S9G, R32L, L/K/Q34A/P, E35D/A, and K/R36A;T/S9G, R33S/N, L/K/Q34A/P, E35D/A, and K/R36A; G/A31D/E, R32L, R33S/N,L/K/Q34A/P, and E35D/A; G/A31D/E, R32L, R33S/N, L/K/Q34A/P, and K/R36A;G/A31D/E, R32L, R33S/N, E35D/A, and K/R36A; G/A31 D/E, R32L, L/K/Q34A/P,E35D/A, and K/R36A; G/A31 D/E, R33S/N, L/K/Q34A/P, E35D/A, and K/R36A;or R32L, R33S/N, L/K/Q34A/P, E35D/A, and K/R36A. In any of theseaspects, the variant may further comprise a deletion at a positioncorresponding to position 10 of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of substitutions atany six positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2. For example, the variant may comprise or consist ofsix substitutions corresponding to positions 9, 31, 32, 33, 34, and 35;9, 31, 32, 33, 34, and 36; 9, 31, 32, 33, 35, and 36; 9, 31, 32, 34, 35,and 36; 9, 31, 33, 34, 35, and 36; 9, 32, 33, 34, 35, and 36; or 31, 32,33, 34, 35, and 36, such as those described above. In one aspect, thevariant comprises or consists of the substitutions T/S9G, G/A31 D/E,R32L, R33S/N, L/K/Q34A/P, and E35D/A; T/S9G, G/A31D/E, R32L, R33S/N,L/K/Q34A/P, and K/R36A; T/S9G, G/A31D/E, R32L, R33S/N, E35D/A, andK/R36A; T/S9G, G/A31D/E, R32L, L/K/Q34A/P, E35D/A, and K/R36A; T/S9G,G/A31 D/E, R33S/N, L/K/Q34A/P, E35D/A, and K/R36A; T/S9G, R32L, R33S/N,L/K/Q34A/P, E35D/A, and K/R36A; or G/A31D/E, R32L, R33S/N, L/K/Q34A/P,E35D/A, and K/R36A. In any of these aspects, the variant may furthercomprise a deletion at a position corresponding to position 10 of SEQ IDNO: 2.

In another aspect, the variant comprises or consists of substitutions atall seven positions corresponding to positions 9, 31, 32, 33, 34, 35 and36 of SEQ ID NO: 2, such as those described above. In one aspect, thevariant comprises or consists of the substitutions T/S9G, G/A31 D/E,R32L, R33S/N, L/K/Q34A/P, E35D/A and K/R36A. In either of these aspects,the variant may further comprise a deletion at a position correspondingto position 10 of SEQ ID NO: 2.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, as described herein, certain amino acid changes are ofsuch a nature that the physico-chemical properties of the polypeptidesare altered. For example, the amino acid changes to positionscorresponding to any of the positions 9, 31, 32, 33, 34, 35 and 36 ofSEQ ID NO: 2 (and an optional deletion at position 10), may altercofactor specificity, such as increasing the specificity for NAD(H)compared to NADP(H).

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for 3-HPDH activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

For example, essential amino acids of SEQ ID NO: 2 can be identified byanalysis of crystallography data described in Yamazawa et al., 2011, J.Biochem. 149(6): 701-712 (see Worldwide Protein Data Bank;http://www.wwpdb.org; PDB codes: 3ASU and 3ASV), wherein the active sitestructure is identified, including the catalytic tetrad at positions106, 134, 147, and 151. Additional amino acid residues important forenzyme activity are identified therein based on site-directedmutagenesis studies. Similarly, essential amino acids of SEQ ID NO: 6can be identified by analysis of available crystallography data (seeWorldwide Protein Data Bank; http://www.wwpdb.org; PDB code: 3RKU). Theidentity of corresponding essential amino acids for SEQ ID NO: 4 can beinferred from an alignment with SEQ ID NO: 2 and SEQ ID NO: 6, as shownin FIG. 2.

In some aspects, the variants consist of at least 185 amino acids, e.g.,at least 200, 210, 220, 230, or 240 amino acids.

In some embodiments, the variant has increased specificity for NAD(H)compared to NADP(H). In some embodiments, the variant has more than2-fold, e.g., more than 5-fold, 10-fold, 20-fold, 50-fold, 100-fold,200-fold, 500-fold, or 1000-fold specificity for NAD(H) compared toNADP(H)

In some embodiments, the variant has one or more improved propertiescompared to the parent, such as improved catalytic efficiency, improvedcatalytic rate, improved chemical stability, improved oxidationstability, improved pH activity, improved pH stability, improvedspecific activity, improved stability under storage conditions, improvedsubstrate binding, improved substrate cleavage, improved substratespecificity, improved substrate stability, improved surface properties,improved thermal activity, and/or improved thermostability.

Parent 3-Hydroxypropionate Dehydrogenases

The parent 3-HPDH may be (a) a polypeptide having at least 60% sequenceidentity to SEQ ID NO: 2, 4, or 6; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with thefull-length complementary strand of SEQ ID NO: 1, 3, or 5; or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to SEQ ID NO: 1, 3, or 5.

In one aspect, the parent 3-HPDH has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 2. In one aspect, the amino acidsequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 2. In another aspect, theparent comprises or consists of the amino acid sequence of SEQ ID NO: 2.In another embodiment, the parent is an allelic variant of SEQ ID NO: 2.In another aspect, the parent is a fragment of SEQ ID NO: 2.

In another aspect, the parent 3-HPDH has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to SEQ ID NO: 4. In one aspect, the aminoacid sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 4. In another aspect, theparent comprises or consists of the amino acid sequence of SEQ ID NO: 4.In another embodiment, the parent is an allelic variant of SEQ ID NO: 4.In another aspect, the parent is a fragment of SEQ ID NO: 4.

In another aspect, the parent 3-HPDH has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to SEQ ID NO: 6. In one aspect, the aminoacid sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10, from SEQ ID NO: 6. In another aspect, theparent comprises or consists of the amino acid sequence of SEQ ID NO: 6.In another embodiment, the parent is an allelic variant of SEQ ID NO: 6.In another aspect, the parent is a fragment of SEQ ID NO: 6.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under at least low stringency conditions, e.g., mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with thefull-length complementary strand of SEQ ID NO: 1. (Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.). In another aspect, the parent is encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with thefull-length complementary strand of SEQ ID NO: 3. In another aspect, theparent is encoded by a polynucleotide that hybridizes under at least lowstringency conditions, e.g., medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with the full-length complementary strand of SEQID NO: 5.

The polynucleotide of SEQ ID NO: 1, 3, 5, or a subsequence thereof, aswell as the polypeptide of SEQ ID NO: 2, 4, 6, or a fragment thereof,may be used to design nucleic acid probes to identify and clone DNAencoding a parent from strains of different genera or species accordingto methods well known in the art. In particular, such probes can be usedfor hybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1, 3, 5, or a subsequence thereof, the carrier materialis used in a Southern blot.

In one aspect, the nucleic acid probe is a polynucleotide having SEQ IDNO: 1, 3, or 5. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 2, 4, 6; or afragment thereof.

In another embodiment, the parent is encoded by a polynucleotide havingat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to SEQ IDNO: 1. In another embodiment, the parent is encoded by a polynucleotidehaving at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 3. In another embodiment, the parent is encoded by apolynucleotide having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 5.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial 3-HPDH. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces 3-HPDH, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma 3-HPDH.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis 3-HPDH.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus 3-HPDH.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans 3-HPDH.

The parent may be a fungal 3-HPDH. For example, the parent may be ayeast 3-HPDH such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, Yarrowia or Issatchenkia 3-HPDH; or a filamentousfungal 3-HPDH such as an Acremonium, Agaricus, Alternaria, Aspergillus,Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria 3-HPDH.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis 3-HPDH.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reese, or Trichoderma viride 3-HPDH.

In one aspect, the parent 3-HPDH is from E. coli, such as the E. coli3-HPDH of SEQ ID NO: 2. In another aspect, the parent 3-HPDH is fromIssatchenkia, such as the Issatchenkia orientalis 3-HPDH of SEQ ID NO:4. In another aspect, the parent 3-HPDH is from Saccharomyces, such asthe Saccharomyces cerevisiae 3-HPDH of SEQ ID NO: 6.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

Also described are methods for obtaining a variant having 3-HPDHactivity, comprising: (a) introducing into a parent 3-HPDH asubstitution at one or more (e.g., two, several) positions correspondingto positions 9, 31, 32, 33, 34, 35, and 36 of SEQ ID NO: 2; and (b)recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used to prepare thevariants described herein. For example, there are many commercial kitsavailable that can be used.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides, Nucleic Acid Constructs, and Expression Vectors

In one aspect are polynucleotides (e.g., isolated polynucleotides)encoding the polypeptides and variants described herein, as well asnucleic acid constructs and expression vectors comprising thepolynucleotides.

The nucleic acid constructs comprise a polynucleotide encoding apolypeptide or variant described herein operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcohol3-hydroxypropionate dehydrogenase/glyceraldehyde-3-phosphate3-hydroxypropionate dehydrogenase (ADH1, ADH2/GAP), Saccharomycescerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiaemetallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglyceratekinase. Other useful promoters for yeast host cells are described byRomanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate 3-hydroxypropionate dehydrogenase. Otheruseful terminators for yeast host cells are described by Romanos et al.,1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcohol 3-hydroxypropionatedehydrogenase/glyceraldehyde-3-phosphate 3-hydroxypropionatedehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Recombinant expression vectors comprise a polynucleotide encoding apolypeptide or variant described herein, a promoter, and transcriptionaland translational stop signals. The various nucleotide and controlsequences may be joined together to produce a recombinant expressionvector that may include one or more convenient restriction sites toallow for insertion or substitution of the polynucleotide encoding thevariant at such sites. Alternatively, the polynucleotide may beexpressed by inserting the polynucleotide or a nucleic acid constructcomprising the polynucleotide into an appropriate vector for expression.In creating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells comprisinga polynucleotide encoding a polypeptide or variant described hereinoperably linked to one or more control sequences that direct production,e.g., for use in an active 3-HP pathway. A construct or vectorcomprising a polynucleotide is introduced into a host cell so that theconstruct or vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication.

In some aspects, a host cell may be selected for the recombinantproduction and recovery of the polypeptide or variant described herein.In other aspects, the host cell comprises an active 3-HP pathway and ischosen to express the polypeptide or variant described herein as a 3-HPpathway gene in the recombinant production of 3-HP by the cell (e.g., asdescribed in WO2012/074818, the content of which is hereby incorporatedby reference). Such cells can produce 3-HP from a fermentable sugar or amalonyl semialdehyde precursor.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK). The fungalhost cell may be a yeast cell. “Yeast” as used herein includesascosporogenous yeast (Endomycetales), basidiosporogenous yeast, andyeast belonging to the Fungi Imperfecti (Blastomycetes). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner, Passmore, and Davenport, editors, Soc.App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

In some aspects, the host cell is selected from Issatchenkia, Candida,Kluyveromyces, Pichia, Schizosaccharomyces, Torulaspora,Zygosaccharomyces, and Saccharomyces. In some aspects, the host cell isa I. orientalis, C. lambica, or S. bulderi host cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

In some aspects, the host cell comprises an active 3-HP pathway whichincludes a polynucleotide that encodes a polypeptide or variantdescribed herein and is capable of producing 3-HP from a fermentablesugar (e.g., glucose) or pyruvate. Active 3-HP pathways such as thepathway shown in FIG. 1 are known in the art (see, for example,WO2012/074818, the content of which is hereby incorporated byreference). The host cell may comprises PEP carboxylase activity orpyruvate carboxylase activity; aspartate aminotransferase activity;aspartate decarboxylase activity; and beta-alanine/alpha-ketoglutarateaminotransferase (BAAT) activity. (see, for example, WO02/42418 andWO2008/027742, the content of which is hereby incorporated byreference). Such enzyme activities may result from endogenous geneexpression, expression of heterologous polynucleotides encoding theenzymes in the metabolic pathway, or from a combination of endogenousgene expression supplemented with expression of one or more (e.g., two,several) heterologous polynucleotides. In some embodiments, the hostcell comprises a heterologous polynucleotide that encodes a PEPcarboxylase, a heterologous polynucleotide that encodes a pyruvatecarboxylase, a heterologous polynucleotide that encodes a aspartateaminotransferase, a heterologous polynucleotide that encodes a aspartatedecarboxylase, and/or a heterologous polynucleotide that encodes a BAAT.

In some aspects, the host cell is a 3-HP resistant host cell. A“3-HP-resistant host cell” as used herein refers to a host cell thatexhibits an average glycolytic rate of at least 2.5 g/L/hr in mediacontaining 75 g/L or greater 3-HP at a pH of less than 4.0. Such ratesand conditions represent an economic process for producing 3-HP. Incertain of these embodiments, the host cells may exhibit 3-HP resistancein their native form. In other embodiments, the cells may have undergonemutation and/or selection before, during, or after introduction ofgenetic modifications related to an active 3-HP fermentation pathway,such that the mutated and/or selected cells possess a higher degree ofresistant to 3-HP than wild-type cells of the same species. In certainembodiments, mutation and/or selection may be carried out on cells thatexhibit 3-HP resistance in their native form. Cells that have undergonemutation and/or selection may be tested for sugar consumption and othercharacteristics in the presence of varying levels of 3-HP in order todetermine their potential as industrial hosts for 3-HP production. Inaddition to 3-HP resistance, the host cells provided herein may haveundergone mutation and/or selection for resistance to one or moreadditional organic acids or to other fermentation products, byproducts,or media components.

Selection for resistance to 3-HP or to other compounds may beaccomplished using methods well known in the art. For example, selectionmay be carried out using a chemostat. A chemostat is a device thatallows for a continuous culture of microorganisms (e.g., yeast) whereinthe specific growth rate and cell number can be controlledindependently. A continuous culture is essentially a flow system ofconstant volume to which medium is added continuously and from whichcontinuous removal of any overflow can occur. Once such a system is inequilibrium, cell number and nutrient status remain constant, and thesystem is in a steady state. A chemostat allows control of both thepopulation density and the specific growth rate of a culture throughdilution rate and alteration of the concentration of a limitingnutrient, such as a carbon or nitrogen source. By altering theconditions as a culture is grown (e.g., decreasing the concentration ofa secondary carbon source necessary to the growth of the inoculumstrain, among others), microorganisms in the population that are capableof growing faster at the altered conditions will be selected and willoutgrow microorganisms that do not function as well under the newconditions. Typically such selection requires the progressive increaseor decrease of at least one culture component over the course of growthof the chemostat culture. The operation of chemostats and their use inthe directed evolution of microorganisms is well known in the art (see,e.g., Novick Proc Natl Acad Sci USA 36:708-719 (1950), Harder J ApplBacteriol 43:1-24 (1977).

In some aspects, the host cell secretes (and/or is capable of secreting)an increased level of 3-HP compared to the host cell without thepolynucleotide that encodes the polypeptide or variant described hereinwhen cultivated under the same conditions. In some embodiments, the hostcell secretes and/or is capable of secreting an increased level of 3-HPof at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least25%, at least 50%, at least 100%, at least 150%, at least 200%, at least300%, or at 500% compared to the host cell without the polynucleotidethat encodes the polypeptide or variant described herein, whencultivated under the same conditions. Examples of suitable cultivationconditions are described below and will be readily apparent to one ofskill in the art based on the teachings herein. In some embodiments, thehost cell produces (and/or is capable of producing) 3-HP at a yield ofat least than 10%, e.g., at least than 20%, at least than 30%, at leastthan 40%, at least than 50%, at least than 60%, at least than 70%, atleast than 80%, or at least than 90%, of theoretical. In someembodiments, the host cell has a 3-HP volumetric productivity greaterthan about 0.1 g/L per hour, e.g., greater than about 0.2 g/L per hour,0.5 g/L per hour, 0.6 g/L per hour, 0.7 g/L per hour, 0.8 g/L per hour,0.9 g/L per hour, 1.0 g/L per hour, 1.1 g/L per hour, 1.2 g/L per hour,1.3 g/L per hour, 1.5 g/L per hour, 1.75 g/L per hour, 2.0 g/L per hour,2.25 g/L per hour, 2.5 g/L per hour, or 3.0 g/L per hour; or betweenabout 0.1 g/L per hour and about 2.0 g/L per hour, e.g., between about0.3 g/L per hour and about 1.7 g/L per hour, about 0.5 g/L per hour andabout 1.5 g/L per hour, about 0.7 g/L per hour and about 1.3 g/L perhour, about 0.8 g/L per hour and about 1.2 g/L per hour, or about 0.9g/L per hour and about 1.1 g/L per hour.

The host cells may be cultivated in a nutrient medium suitable forproduction of the polypeptides and variants described herein usingmethods well known in the art. For example, the cell may be cultivatedby shake flask cultivation, and small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in a suitable mediumand under conditions allowing the desired polypeptide to be expressedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, asdescribed herein, using procedures known in the art. Suitable media areavailable from commercial suppliers, may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection), or may be prepared from commercially available ingredients.

As described supra, enzyme activities of the enzymes described hereincan be detected using methods known in the art. These detection methodsmay include use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. See, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Third Ed., Cold SpringHarbor Laboratory, New York (2001); Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999); and Hanaiet al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).

Methods of Production

The present invention also relates to methods of producing a polypeptideor variant described herein, comprising: (a) cultivating a host cellcomprising a polynucleotide encoding the polypeptide or variant underconditions suitable for expression; and (b) recovering the polypeptideor variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide or variant described herein using methodsknown in the art. For example, the cell may be cultivated by shake flaskcultivation, or small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the variant to be expressed and/or isolated.The cultivation takes place in a suitable nutrient medium comprisingcarbon and nitrogen sources and inorganic salts, using procedures knownin the art. Suitable media are available from commercial suppliers ormay be prepared according to published compositions (e.g., in cataloguesof the American Type Culture Collection). If the variant is secretedinto the nutrient medium, the variant can be recovered directly from themedium. If the variant is not secreted, it can be recovered from celllysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assay,such as the assays described in the Examples section, may be used todetermine the enzymatic activity.

The polypeptide or variant may be recovered using methods known in theart. For example, the variant may be recovered from the nutrient mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide or variant may be purified by a variety of proceduresknown in the art including, but not limited to, chromatography (e.g.,ion exchange, affinity, hydrophobic, chromatofocusing, and sizeexclusion), electrophoretic procedures (e.g., preparative isoelectricfocusing), differential solubility (e.g., ammonium sulfateprecipitation), SDS-PAGE, or extraction (see, e.g., ProteinPurification, Janson and Ryden, editors, VCH Publishers, New York, 1989)to obtain substantially pure variants.

In an alternative aspect, the polypeptide or variant is not recovered,but rather a host cell expressing the polypeptide is used as part of ametabolic pathway, as described herein for the production of 3-HP.

Methods of Producing 3-HP

The host cells described herein may be used for the production of 3-HP.In one aspect is a method of producing 3-HP from a fermentable sugar(e.g., glucose) or pyruvate, comprising: (a) cultivating any one of thehost cells described herein (e.g., a host cell that comprises an active3-HP pathway and a polynucleotide that encodes a 3-HPDH describedherein) in a medium under suitable conditions to produce the 3-HP; and(b) recovering the 3-HP. In some embodiments of the method, the hostcell further comprises PEP carboxylase activity or pyruvate carboxylaseactivity; aspartate aminotransferase activity; aspartate decarboxylaseactivity; and beta-alanine/alpha-ketoglutarate aminotransferase (BAAT)activity. In some embodiments, the host cell comprises a heterologouspolynucleotide that encodes a PEP carboxylase, a heterologouspolynucleotide that encodes a pyruvate carboxylase, a heterologouspolynucleotide that encodes a aspartate aminotransferase, a heterologouspolynucleotide that encodes a aspartate decarboxylase, and/or aheterologous polynucleotide that encodes a BAAT. In some embodiments ofthe methods, the host cells are 3-HP resistant host cells, as describedsupra.

In one aspect is a method of producing 3-HP from a fermentable sugar(e.g., glucose) or pyruvate, comprising: (a) cultivating any one of thehost cells described herein (e.g., a host cell that comprises an active3-HP pathway and a polynucleotide that encodes a 3-HPDH variant of SEQID NO: 2, 4, or 6 described herein) in a medium under suitableconditions to produce the 3-HP; and (b) recovering the 3-HP. In someembodiments of the method, the 3-HPDH variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to SEQ ID NO: 2, 4, or 6. In someembodiments, the host cell further comprises PEP carboxylase activity orpyruvate carboxylase activity; aspartate aminotransferase activity;aspartate decarboxylase activity; and beta-alanine/alpha-ketoglutarateaminotransferase (BAAT) activity. In some embodiments, the host cellcomprises a heterologous polynucleotide that encodes a PEP carboxylase,a heterologous polynucleotide that encodes a pyruvate carboxylase, aheterologous polynucleotide that encodes a aspartate aminotransferase, aheterologous polynucleotide that encodes a aspartate decarboxylase,and/or a heterologous polynucleotide that encodes a BAAT. In someembodiments of the methods, the host cells are 3-HP resistant hostcells, as described supra.

In one aspect is a method of producing 3-HP from a fermentable sugar(e.g., glucose) or pyruvate, comprising: (a) cultivating any one of thehost cells described herein (e.g., a host cell that comprises an active3-HP pathway and a polynucleotide that encodes a 3-HPDH related to SEQID NO: 82) in a medium under suitable conditions to produce the 3-HP;and (b) recovering the 3-HP. In some embodiments of the method, the3-HPDH has at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to theP. putida 3-HPDH of SEQ ID NO: 82. In some embodiments, the host cellfurther comprises PEP carboxylase activity or pyruvate carboxylaseactivity; aspartate aminotransferase activity; aspartate decarboxylaseactivity; and beta-alanine/alpha-ketoglutarate aminotransferase (BAAT)activity. In some embodiments, the host cell comprises a heterologouspolynucleotide that encodes a PEP carboxylase, a heterologouspolynucleotide that encodes a pyruvate carboxylase, a heterologouspolynucleotide that encodes an aspartate aminotransferase, aheterologous polynucleotide that encodes a aspartate decarboxylase,and/or a heterologous polynucleotide that encodes a BAAT. In someembodiments of the methods, the host cells are 3-HP resistant hostcells, as described supra.

Methods for the production of 3-HP may be performed in a fermentablemedium comprising any one or more (e.g., two, several) sugars, such asglucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. In some instances,the fermentation medium is derived from a natural source, such as sugarcane, starch, or cellulose, and may be the result of pretreating thesource by enzymatic hydrolysis (saccharification).

In addition to the appropriate carbon sources from one or more (e.g.,two, several) sugar(s), the fermentable medium may contain othernutrients or stimulators known to those skilled in the art, such asmacronutrients (e.g., nitrogen sources) and micronutrients (e.g.,vitamins, mineral salts, and metallic cofactors). In some aspects, thecarbon source can be preferentially supplied with at least one nitrogensource, such as yeast extract, N₂, peptone (e.g., Bacto™ Peptone), orsoytone (e.g., Bacto™ Soytone). Nonlimiting examples of vitamins includemultivitamins, biotin, pantothenate, nicotinic acid, meso-inositol,thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin,and Vitamins A, B, C, D, and E. Examples of mineral salts and metalliccofactors include, but are not limited to Na, P, K, Mg, S, Ca, Fe, Zn,Mn, and Cu.

Suitable conditions used for the methods of 3-HP production may bedetermined by one skilled in the art in light of the teachings herein.In some aspects of the methods, the host cells are cultivated for about12 hours to about 216 hours, such as about 24 hours to about 144 hours,or about 36 hours to about 96 hours. The temperature is typicallybetween about 26° C. to about 60° C., e.g., about 34° C. to about 50°C., and at a pH of about 3.0 to about 8.0, such as about 3.0 to about7.0, about 3.0 to about 6.0, about 3.0 to about 5.0, about 3.5 to about4.5, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about6.0, about 4.0 to about 5.0, about 5.0 to about 8.0, about 5.0 to about7.0, or about 5.0 to about 6.0. In some aspects of the methods, theresulting intracellular pH of the host cell is about 3.0 to about 8.0,such as about 3.0 to about 7.0, about 3.0 to about 6.0, about 3.0 toabout 5.0, about 3.5 to about 4.5, about 4.0 to about 8.0, about 4.0 toabout 7.0, about 4.0 to about 6.0, about 4.0 to about 5.0, about 5.0 toabout 8.0, about 5.0 to about 7.0, or about 5.0 to about 6.0.Cultivation may be performed under anaerobic, microaerobic, or aerobicconditions, as appropriate. In some aspects, the cultivation isperformed under anaerobic conditions. Suitable buffering agents areknown in the art.

Cultivation may be performed under anaerobic, substantially anaerobic(microaerobic), or aerobic conditions, as appropriate. Briefly,anaerobic refers to an environment devoid of oxygen, substantiallyanaerobic (microaerobic) refers to an environment in which theconcentration of oxygen is less than air, and aerobic refers to anenvironment wherein the oxygen concentration is approximately equal toor greater than that of the air. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains less than 10% of saturation. Substantially anaerobic conditionsalso includes growing or resting cells in liquid medium or on solid agarinside a sealed chamber maintained with an atmosphere of less than 1%oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases. In some embodiments, the cultivation is performed underanaerobic conditions or substantially anaerobic conditions.

The methods of described herein can employ any suitable fermentationoperation mode. For example, a batch mode fermentation may be used witha close system where culture media and host microorganism, set at thebeginning of fermentation, have no additional input except for thereagents certain reagents, e.g., for pH control, foam control or othersrequired for process sustenance. The process described herein can alsobe employed in Fed-batch or continuous mode.

The methods described herein may be practiced in several bioreactorconfigurations, such as stirred tank, bubble column, airlift reactor andothers known to those skilled in the art.

The methods may be performed in free cell culture or in immobilized cellculture as appropriate. Any material support for immobilized cellculture may be used, such as alginates, fibrous bed, or argyle materialssuch as chrysotile, montmorillonite KSF and montmorillonite K-10.

In one aspect of the methods, the 3-HP is produced at a titer greaterthan about 10 g/L, e.g., greater than about 25 g/L, 50 g/L, 75 g/L, 100g/L, 125 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, 210g/L, 225 g/L, 250 g/L, 275 g/L, 300 g/L, 325 g/L, 350 g/L, 400 g/L, or500 g/L; or between about 10 g/L and about 500 g/L, e.g., between about50 g/L and about 350 g/L, about 100 g/L and about 300 g/L, about 150 g/Land about 250 g/L, about 175 g/L and about 225 g/L, or about 190 g/L andabout 210 g/L. In one aspect of the methods, the 3-HP is produced at atiter greater than about 0.01 gram per gram of carbohydrate, e.g.,greater than about 0.02, 0.05, 0.75, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, or 1.0 gram per gram of carbohydrate.

In one aspect of the methods, the amount of produced 3-HP is at least5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 50%, or at least 100% greater compared tocultivating the host cell without the polynucleotide that encodes a3-HPDH under the same conditions.

The recombinant 3-HP can be optionally recovered and purified from thefermentation medium using any procedure known in the art including, butnot limited to, chromatography (e.g., size exclusion chromatography,adsorption chromatography, ion exchange chromatography), electrophoreticprocedures, differential solubility, distillation, extraction (e.g.,liquid-liquid extraction), pervaporation, extractive filtration,membrane filtration, membrane separation, reverse osmosis,ultrafiltration, or crystallization.

In some aspects of the methods, the recombinant 3-HP before and/or afterbeing optionally purified is substantially pure. With respect to themethods of producing 3-HP, “substantially pure” intends a recoveredpreparation of 3-HP that contains no more than 15% impurity, whereinimpurity intends compounds other than 3-HP. In one variation, apreparation of substantially pure 3-HP is provided wherein thepreparation contains no more than 25% impurity, or no more than 20%impurity, or no more than 10% impurity, or no more than 5% impurity, orno more than 3% impurity, or no more than 1% impurity, or no more than0.5% impurity.

Suitable assays to test for the production of 3-HP for the methods ofproduction and host cells described herein can be performed usingmethods known in the art. For example, the final 3-HP (and other organiccompounds) can be analyzed by methods such as HPLC (High PerformanceLiquid Chromatography), GC-MS (Gas Chromatography Mass Spectroscopy) andLC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitableanalytical methods using routine procedures well known in the art. Therelease of 3-HP in the fermentation broth can also be tested with theculture supernatant. Byproducts and residual sugar in the fermentationmedium (e.g., glucose) can be quantified by HPLC using, for example, arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)),or using other suitable assay and detection methods well known in theart.

Plants

Also described are plants, e.g., transgenic plants, plant parts, orplant cells, comprising a polynucleotide described herein so as toexpress and produce the polypeptides and variants described herein inrecoverable quantities.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

In one aspect is a method of producing a polypeptide or variantdescribed herein comprising: (a) cultivating a transgenic plant or aplant cell comprising a polynucleotide encoding the polypeptide orvariant under conditions conducive for production; and (b) recoveringthe polypeptide or variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

The E. coli strain MG1655 (NN059268) was used as the source of DNAencoding the ydfG 3-HPDH gene. Strains MG1655, SoloPack Gold (AgilentTechnologies, Inc., Santa Clara, Calif., USA), and SURE cells (AgilentTechnologies, Inc.) were used to express the ydfG plasmids.

The I. orientalis strain MBin500 was used as the source of DNA encodingthe I. orientalis YMR226c 3-HPDH gene, as described in WO2012/074818. I.orientalis strain McTs259 (WO2012/074818) was used to express thedescribed 3-HPDH genes for 3-HP production.

Media

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

2XYT plates were composed of 16 g of tryptone, 10 g of yeast extract, 5g of NaCl, 15 g of Bacto agar, and deionized water to 1 liter.

TABLE 0 Primer Sequences Identifier SEQ ID Sequence (5′-3′) 000001 15CGGAATTCATGATCGTTTTAGTAACTGGAGC 000002 16 CGGGATCCTTACTGACGGTGGACATTCAG614464 34 TCGCCACTGATCTGAACCCGGAAGCGTTGCAGGAGTTAAAAGA 614465 35TCTTTTAACTCCTGCAACGCTTCCGGGTTCAGATCAGTGGCGA 614466 36CCACTGATCTGAACCCGGCCCGGTTGCAGGAGTTAAAAGACGA 614467 37TCGTCTTTTAACTCCTGCAACCGGGCCGGGTTCAGATCAGTGG 614468 38GGCATAAAGTTATCGCCACTGGCCTGAACCCGGCCGCGTTGCA 614469 39TGCAACGCGGCCGGGTTCAGGCCAGTGGCGATAACTTTATGCC 614470 40TCGTTTTAGTAACTGGAGCAACGGCAGGTTTTGGTGAATGCATT 614471 41AATGCATTCACCAAAACCTGCCGTTGCTCCAGTTACTAAAACGA 614472 42ATAAAGTTATCGCCACTGATCGTAACCCGGCCGCGTTGCAGGA 614473 43TCCTGCAACGCGGCCGGGTTACGATCAGTGGCGATAACTTTAT 614476 44AACTCCTGCAACGCGGCCGGGCGCAGATCAGTGGCGATAACTT 614477 45AAGTTATCGCCACTGATCTGCGCCCGGCCGCGTTGCAGGAGTT 614479 46TTATCGCCACTGATCTGAACCAGGCCGCGTTGCAGGAGTTAAA 614480 47TTTAACTCCTGCAACGCGGCCTGGTTCAGATCAGTGGCGATAA 614546 48TAAAGTTATCGCCACTGATCTGCGCCCGGAAGCGTTGCAGGAGTTAAAA GACG 614547 49CGTCTTTTAACTCCTGCAACGCTTCCGGGCGCAGATCAGTGGCGATAAC TTTA 614548 50AGTTATCGCCACTGATCTGCGCCAGGCCGCGTTGCAGGAGTTAAAAGAC GAAC 614549 51GTTCGTCTTTTAACTCCTGCAACGCGGCCTGGCGCAGATCAGTGGCGAT AACT 614550 52GCATAAAGTTATCGCCACTGATCTGCGCCAGGAAGCGTTGCAGGAGTTA AAAGACGAAC 614551 53GTTCGTCTTTTAACTCCTGCAACGCTTCCTGGCGCAGATCAGTGGCGAT AACTTTATGC 614552 54GCATAAAGTTATCGCCACTGATCTGAACCAGGAAGCGTTGCAGGAGTTA AAAG 614553 55CTTTTAACTCCTGCAACGCTTCCTGGTTCAGATCAGTGGCGATAACTTTA TGC 614697 56GTCATCGTAGTCTAGATAAAATGATCGTTTTGGTCACCGG 614698 57GTGCTCCATTAATTAATTATTGTCTGTG 614967 58 GCGGAATTCATGTTTGGTAATATTTCCCAA614968 59 GATCCCGGGCTATTTATCTAATGATCCTC 614973 60GTTTTAGTAACTGGAGCAGGCGCAGGTTTTGGTGAATGC 614974 61GCATTCACCAAAACCTGCGCCTGCTCCAGTTACTAAAAC 614975 62CATAAAGTTATCGCCACTGATCGTCGCCAGGAACGGTTG 614976 63CAACCGTTCCTGGCGACGATCAGTGGCGATAACTTTATG 614977 64TAAAGTTATCGCCACTGATCGTCGCCAGGAAGCGTTGCAG 614978 65CTGCAACGCTTCCTGGCGACGATCAGTGGCGATAACTTTA 614979 66ACTGATCTGCGCCAGGAACGGTTGCAGGAGTTAAAAGAC 614980 67GTCTTTTAACTCCTGCAACCGTTCCTGGCGCAGATCAGT 615004 68ATCCTAATTACAGGTGCGGGTACTGGTATCGGATACCAT 615005 69ATGGTATCCGATACCAGTACCCGCACCTGTAATTAGGAT 615006 70TTGAAGTTGGTTTTGGCTGATTTAAGAAAGGAGAAGCTGGAG 615007 71CTCCAGCTTCTCCTTTCTTAAATCAGCCAAAACCAACTTCAA 615428 72TTGCAGGCAAGAACATCCTAATTACAGGTGC 615429 73GCACCTGTAATTAGGATGTTCTTGCCTGCAA 615485 74GTAGCTAGCTAAAATGTTTGGTAATATTTCCCA 615486 75TGCTTAATTAACTATTTATCTAATGATCCTC 615890 76TTGGTCACCGGTGCAGGTGCAGGTTTCGGCGAA 615891 77TTCGCCGAAACCTGCACCTGCACCGGTGACCAA 615892 78ACAAGGTTATCGCTACCGACTTGAGACAAGAGAGATTGCA 615893 79TGCAATCTCTCTTGTCTCAAGTCGGTAGCGATAACCTTGT

Example 1 Construction of an Expression Vector for the E. Coli ydfG3-HPDH Gene

The E. coli ydfG 3-HPDH coding sequence was amplified by PCR using twosynthetic oligonucleotide primers designed to generate an EcoRIrestriction site at the 5′ end and a BamHI restriction site at the 3′end for integration into pTrc99A (FIG. 4; see Amann, E., et al. (1988).“Tightly regulated tac promoter vectors useful for the expression ofunfused and fused proteins in Escherichia coli.” Gene 69(2): 301-315).

E. coli genomic DNA for PCR was obtained by isolating a single colony ofE. coli MG1655 from a 2XYT plate and dissolving into 25 μl 1% TritonX-100, 20 mM Tris pH 8.5, 2 mM EDTA (CLS solution), heated at 80° C. for10 minutes and then cooled on ice. Three microliters of this solutionused as a template in a PCR reaction further containing 1× PfxAmplification buffer, fifty picomoles each of primers 000001 and 000002,0.2 mM each of dATP, dGTP, dCTP, and dTTP, and 2.5 units Platinum® PfxDNA Polymerase (Invitrogen, Carlsbad, Calif., USA) in a final volume of50 μl. The amplification reaction was performed in an EPPENDORF®MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for one cycle at 95° C. for 2 minutes; and 25 cycles each at95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute.After the 25 cycles, the reaction was incubated at 72° C. for 3 minutesand then cooled at 10° C. until further processed.

Five microliters of the PCR reaction mixture was subjected to 1%TBE-agarose gel electrophoresis with ethidium bromide in TBE buffer toidentify the desired 765 bp PCR fragment. The PCR fragment from theremaining 45 μl of the PCR reaction mixture was purified using aQIAquick PCR Purification Kit (Qiagen Inc., Valencia, Calif., USA). Thepurified fragment was digested with EcoRI and BamHI (New EnglandBiolabs, Ipswich, Mass., USA) and analyzed on a 1% TBE-agarose gel withethidium bromide.

The plasmid pTrc99A (supra) was digested with EcoRI and BamHI, and theresulting fragments separated by 1% TBE-agarose gel electrophoresisfollowed by visualization with a DARK READER™ (Clare Chemical Research,Dolores, Colo., USA). The desired 4.1 kb fragment was excised from thegel with a disposable razor blade and purified using a QIAquick GelExtraction Kit (Qiagen, Inc.).

Cloning of the DNA fragment containing E. coli ydfG into pTrc99A wasperformed using T4 DNA ligase (New England Biolabs). The reactionmixture contained 1× T4 DNA ligase buffer, 1 μl T4 DNA ligase, 1 μl ofthe pTrc99A EcoRI/BamHI digested DNA fragment above, and 5 μl of theydfG EcoRI/BamHI digested PCR product above in total volume of 10 μl.The reaction mixture was incubated at 16° C. overnight and subsequentlyused to transform SURE competent cells (Agilent Technologies, Inc.)according to manufacturer's instructions. After a recovery period, two100 μl aliquots from the transformation mixture were plated onto 150 mm2XYT plates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C.

Recombinant colonies of the transformations were each inoculated into 3ml of LB medium supplemented with ampicillin (100 μg/ml). Plasmid DNAwas prepared from these cultures using a BIOROBOT® 9600 workstation(Qiagen, Inc.) and analyzed by 1% TBE-agarose gel electrophoresisfollowing EcoRI/BamHI digestion. The plasmid DNA from one clonedesignated pMeJi9 and having the correct restriction digest pattern wasfurther subjected to sequence analysis to confirm integration of thecorrect ydfG coding sequence.

Example 2 Construction of E. coli ydfG 3-HPDH Gene Variants

Synthetic DNA sequences encoding the desired ydfG variants andcontaining a 5′ flanking EcoRI restriction site and 3′ Nael restrictionsite were provided in plasmid constructs from DNA2.0 (Menlo Park,Calif., USA). Each plasmid was digested with EcoRI and Nael restrictionenzymes, and the resulting fragments separated on a 1% TAE agarose gelfollowed by visualization with the aid of a DARK READER™ (Clare ChemicalResearch). The desired DNA band containing the ydfG variant encodingsequence was excised from the gel with a disposable razor blade andpurified using NucleoSpin Extract II Kit (Machery-Nagel, Duren,Germany).

Plasmid pMeJi9 (supra) was linearized by digestion with EcoRI and Nael,followed by incubation with Alkaline Phosphatase, Calf Intestinal (CIP)(New England Biolabs) for removal of the 5′ phosphate. The resultingmixture was subjected to gel electrophoresis, visualized, and purifiedas described above to provide the desired 4657 bp DNA fragment.

Cloning of each ydfG variant encoding sequence into linearized pMeJi9was performed by incubating 1× T4 DNA ligase buffer, 1 μl T4 DNA ligase(New England Biolabs), 2 μl EcoRI/Nael linearized pMeJi9, and 15 μl ofthe selected EcoRI/Nael ydfG variant encoding sequence (in total volumeof 20 μl) for 2 hours at room temperature. A 10 μl sample of theincubation reaction was used to transform SoloPack® Gold chemicallycompetent cells according to according to the manufacturer'sinstructions. After a recovery period, two 100 μl aliquots from thetransformation mixture were plated onto 150 mm 2XYT plates supplementedwith 100 μg of ampicillin per ml and incubated overnight at 37° C.Putative recombinant clones were selected from the selection plates andplasmid DNA was prepared from each one using a BIOROBOT® 9600workstation. Clones were analyzed by sequencing. Those plasmids with thecorrect sequence are shown in Table 1.

TABLE 1 Cloning Variant SEQ Plasmid Name ID Name Amino Acid Changes Mut17 pMcTs68 T9G/A10*/G31D/R32L/R33N/Q34P/E35A/R36A Mut2 8 pMcTs69T9G/G31E/R32L/R33N/Q34P/E35A/R36A Mut3 9 pMcTs70T9G/A10*/G31E/R32L/R33N/Q34P/E35A/R36A Mut4 10 pMcTs71T9G/G31D/R32L/R33S/Q34A/E35D/R36A Mut5 11 pMcTs72T9G/A10*/G31D/R32L/R33S/Q34A/E35D/R36A Mut6 12 pMcTs73T9G/G31D/R32L/R33N/Q34P/E35A/R36A Mut7 13 pMcTs74T9G/A10*/G31E/R32L/R33S/Q34A/E35D/R36A Mut8 14 pMcTs75T9G/G31E/R32L/R33S/Q34A/E35D/R36A *represents deletion of the aminoacid.

Additional variants shown in Table 2 below were constructed usingsite-directed mutagenesis and the indicated primers in a PCR reactionusing QuikChange II XL Site-Directed Mutagenesis Kit (AgilentTechnologies, Inc.). The PCR reaction contained 1× Reaction Buffer, 125ng of each primer, 30 ng plasmid DNA template, 1× dNTPs, 1× Quicksolution, 2.5 U PfuUltra HF DNA polymerase in a final volume of 50 μl.The amplification reaction was performed in an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc.) programmed for one cycle at 95° C. for3 minutes; and 18 cycles each at 95° C. for 50 seconds, 60° C. for 50seconds, and 68° C. for 6 minutes. After the 18 cycles, the reaction wasincubated at 68° C. for 7 minutes and then cooled at 10° C. untilfurther processed. To the each PCR reaction 1 μl of DpnI was added andincubated at 37° C. for 1.5 hours to digest template plasmid DNA.

From each site directed PCR reaction, 2.5 μl of the reaction wastransformed into XL10 Gold Super competent cells (Agilent Technologies,Inc.) according to manufacturer's instructions. After a recovery period,two 100 μl aliquots from the transformation mixture were plated onto 150mm 2XYT plates supplemented with 100 μg of ampicillin per ml andincubated overnight at 37° C.

Recombinant colonies of the transformations were each inoculated into 3ml of LB medium supplemented with ampicillin (100 μg/ml). Plasmid DNAwas prepared from these cultures using a BIOROBOT® 9600 workstation(Qiagen, Inc.) and subjected to sequence analysis to confirm the sitedirected mutation in the ydfG coding sequence.

TABLE 2 Cloning Variant SEQ Plasmid Forward Reverse Name ID Name AminoAcid Changes Primer Primer Template Mut9 17 pMcTs79G31D/R32L/R33N/Q34P/E35A/R36A 614470 614471 pMcTs73 Mut10 18 pMcTs80T9G/R32L/R33N/Q34P/E35A/R36A 614468 614469 pMcTs73 Mut11 19 pMcTs81T9G/G31D/R33N/Q34P/E35A/R36A 614472 614473 pMcTs73 Mut12 20 pMcTs82T9G/G31D/R32L/Q34P/E35A/R36A 614476 614477 pMcTs73 Mut13 21 pMcTs83T9G/G31D/R32L/R33N/E35A/R36A 614479 614480 pMcTs73 Mut14 22 pMcTs84T9G/G31D/R32L/R33N/Q34P/R36A 614464 614465 pMcTs73 Mut15 23 pMcTs85T9G/G31D/R32L/R33N/Q34P/E35A 614466 614467 pMcTs73 Mut16 24 pMcTs89T9G/G31D/R32L/R36A 614550 614551 pMcTs73 Mut17 25 pMcTs86T9G/G31D/R32L/E35A/R36A 614548 614549 pMcTs73 Mut18 26 pMcTs87T9G/G31D/R32L/Q34P/R36A 614546 614547 pMcTs73 Mut19 27 pMcTs88T9G/G31D/R32L/R33N/R36A 614552 614553 pMcTs73 Mut20 28 pMcTs98 G31D614975 614976 pMeJi9 Mut21 29 pMcTs99 T9G/G31D/R36A 614977 614978pMcTs89 Mut22 30 pMcTs100 T9G/G31D/R32L 614979 614980 pMcTs89 Mut23 31pMcTs104 T9G 614973 614974 pMeJi9 Mut24 32 pMcTs105 T9G/G31D 614973614974 pMcTs98 Mut25 33 pMcTs114 G31D/R32L 614470 614471 pMcTs100

Example 3 Expression of E. coli ydfG 3-HPDH Gene Variants in MG1655Cells

Electrocompetent MG1655 cells were transformed with the resultingcloning plasmids from Example 2 (or controls pMeJi9 or pTrc99A)according to the procedure described in Sheen, J. (1989).“High-Efficiency Transformation by Electroporation.” Current Protocolsin Molecular Biology. 1.8.4. After a recovery period, two 100 μlaliquots from the transformation reaction were plated onto 150 mm 2XYTplates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C. For each transformation, plasmid DNA of a selectedrecombinant clone was prepared using a BIOROBOT® 9600 workstation andanalyzed by sequencing. The selected clone was then inoculated into aculture of 3 ml of LB media supplemented with 100 μg of ampicillin andincubated overnight at 37° C. with shaking. 250 μl of the overnightculture was added to 25 ml of LB media supplemented with 100 μg ofampicillin per ml in a 125 ml baffled shake flask and grown to OD₆₀₀˜0.6before adding 0.5 mM IPTG to induce expression from the plasmid. After 1hour incubation with IPTG the culture was collected by centrifugationand submitted for enzyme assays, as described below. Samples from thecultures were also collected for SDS-PAGE analysis on an 8-16% Bio-RadCriterion stain-free Tris-HCl gel (Bio-Rad Laboratories, Inc., Hercules,Calif., USA).

Example 4 Cofactor Specificity of Cells Expressing E. coli ydfG 3-HPDHGene Variants

Cultures from Example 3 were harvested by centrifugation (15,000×g at 4°C. for 10 min) and stored at −80° C. Cells were thawed on ice and thepellet was resuspended in Phosphate Buffered Saline (PBS; NaCl, 137 mM;KCl, 2.7 mM; Na₂HPO₄, 10 mM; KH₂PO₄, 1.76 mM) at pH 7.4 containing onetablet of Roche Complete Mini proteases inhibitor cocktail (Roche,Basel) per 10 mL of buffer. Cells were washed three times, and thenresuspended in PBS plus protease inhibitor supplemented with lysozyme(Sigma-Aldrich, Saint-Louis, Mo.) at a concentration of 2 mg/mL. Cellswere then incubated on ice for 30 minutes to allow release ofcytoplasmic content, and membrane debris was collected by centrifugation(15,000×g at 4° C. for 30 min). The supernatant containing the crudeextract (CCE) was transferred to a new tube and kept on ice untilfurther use. CCE protein was quantitated using a Pierce BCA proteindetection kit (Thermo Fisher scientific, Rockford, Ill., USA) using BSAas a standard by following the manufacturer recommendations. Theindicated variants were assayed from the CCE using one or both of theprotocols described below.

A reverse serine dehydrogenase activity assay was conducted with eitherNADP+ or NAD+ cofactor by measuring the appearance over time of theassociated reduced cofactor at 340 nm. The assay was performed in a 96well micro-plate, and the final volume was 300 μL. The reaction wasstarted by adding 30 μL of CCE (supra) into 270 μL of assay buffer (100mM Tris pH 8.0, 10 mM NaHCO₃, 5 mM MgCl₂, 400 mM L-serine and 2 mM ofeither NAD+ or NADP+). Absorbance at 340 nm was followed on amicro-plate reader (Spectra Max 340PC, Molecular Devices LLC, Sunnyvale,Calif., USA) for 10 minutes at room temperature (˜25° C.). One unit wasdefined as the amount of enzyme necessary to produce 1 μmol of eitherNADH or NADPH in one minute in the presence of L-serine at pH 8.0, 25°C.

The results using the serine dehydrogenase assay (see Table 3) showincreased specificity of NAD(H) over to NADP(H) for certaindehydrogenase variants compared to the parent E. coli ydfG gene product(expressed from pMeJi9) and a control lacking a ydfG gene product (blankexpression vector pTrc99A).

TABLE 3 Cloning Serine DeH SA Plasmid (uts/mg prot) NAD+/ Name SEQ IDName NADP+ NAD+ NADP+ Control (ydfG) 1 pMeJi9 12.74 0.35 0.03 Control —pTrc99A 0.35 1.01 2.88 Mut1 7 pMcTs68 0.75 0.58 0.78 Mut2 8 pMcTs69 0.982.69 2.76 Mut3 9 pMcTs70 0.28 0.34 1.21 Mut4 10 pMcTs71 0.88 9.41 10.70Mut5 11 pMcTs72 0.74 0.49 0.66 Mut6 12 pMcTs73 0.80 17.48 21.85 Mut7 13pMcTs74 0.47 0.92 1.97 Mut8 14 pMcTs75 0.97 1.18 1.22

A forward malonate semi-aldehyde reductase assay was conducted bymeasuring the disappearance of either NADH or NADPH over time at 340 nm.Malonate semi-aldehyde was synthesized in-house according to theprotocol developed by Yamada and Jacoby (1960) “Direct conversion ofmalonic semialdehyde to acetyl-coenzyme A”, J. Biol. Chem., 235(3):589-594. The assay was performed in a 96 well micro-plate, and the finalvolume was 200 μL. The reaction was started by adding 30 μL of CCE(supra) into 170 μL of assay buffer (2 mM malonate semialdehyde, 100 mMTris pH 8.0 and 0.5 mM either NADH or NADPH). Absorbance at 340 nm wasfollowed on a micro-plate reader (Spectra Max 340PC, Molecular DevicesLLC) for 10 minutes at room temperature (˜25° C.). One unit was definedas the amount of enzyme necessary to oxidize 1 μmol of either NADH orNADPH in one minute in the presence of malonate semialdehyde at pH 8.0,25° C.

The results using the malonate semi-aldehyde reductase assay (see Table4) show increased specificity of NAD(H) over to NADP(H) for certain3-HPDH variants compared to the parent E. coli ydfG gene product(expressed from pMeJi9) and a control lacking a ydfG gene product (blankexpression vector pTrc99A).

TABLE 4 Cloning Serine DeH SA Plasmid (uts/mg prot) NADH/ Name SEQ IDName NADPH NADH NADPH Control — pTrc99A 4.30 2.75 0.64 Control (ydfG) 1pMeJi9 5174.76 0.00 0.00 Mut1 7 pMcTs68 2.18 0.07 0.03 Mut2 8 pMcTs694.67 0.00 0.00 Mut3 9 pMcTs70 0.00 0.00 0.00 Mut4 10 pMcTs71 2.75 2.871.04 Mut5 11 pMcTs72 4.36 0.00 0.00 Mut6 12 pMcTs73 22.26 39.25 1.76Mut7 13 pMcTs74 0.75 0.00 0.00 Mut8 14 pMcTs75 3.29 0.00 0.00 Mut9 17pMcTs79 3.29 0.00 0.00 Mut10 18 pMcTs80 2.63 0.00 0.00 Mut11 19 pMcTs811.29 0.00 0.00 Mut12 20 pMcTs82 1.40 31.98 22.84 Mut13 21 pMcTs83 2.1614.23 6.60 Mut14 22 pMcTs84 2.00 9.41 4.71 Mut15 23 pMcTs85 2.30 20.669.00 Mut16 24 pMcTs89 0.44 24.33 54.83 Mut17 25 pMcTs86 0.80 27.45 34.17Mut18 26 pMcTs87 1.04 22.34 21.47 Mut19 27 pMcTs88 2.27 15.15 6.67 Mut2028 pMcTs98 0.00 0.00 0.00 Mut21 29 pMcTs99 0.41 5.05 12.45 Mut22 30pMcTs100 1.55 71.91 46.31 Mut23 31 pMcTs104 288.03 0.00 0.00 Mut24 32pMcTs105 0.00 10.56 — Mut25 33 pMcTs114 2.56 0.00 0.00

Example 5 Construction of an Expression Vector for the I. OrientalisYMR226c 3-HPDH Gene

The plasmid pMBin190 (WO2012/074818) contains the I. orientalis YMR226cnucleotide sequence encoding the 3-HPDH of SEQ ID NO: 4 flanked byNheI/PacI sites. The pMBin190 plasmid was digested with NheI and PacI,gel isolated and purified using Qiagen Gel Extraction kit (Qiagen, Inc.)and the 827 bp fragment was ligated into a 7942 bp fragment of pMIBa107(WO2012/074818) digested with XbaI and PacI that was gel isolated andpurified using the Qiagen Gel Extraction kit. Cloning of the DNAfragment containing I. orientalis YMR226c polynucleotide into pMIBa107was performed using T4 DNA ligase (New England Biolabs). The reactionmixture contained 1× T4 DNA ligase buffer, 1 μl T4 DNA ligase, 1 μl ofthe pMIBa107 XbaI/PacI digested DNA fragment above, and 5 μl of theYMR226c NheI/PacI digested product above in total volume of 10 μl. Thereaction mixture was incubated at room temperature for at least 1 hourand subsequently used to transform One Shot TOP10 cells (Invitrogen)according to manufacturer's instructions. After a recovery period, two100 μl aliquots from the transformation mixture were plated onto 150 mm2XYT plates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C. Recombinant colonies of the transformations wereeach inoculated into 3 ml of LB medium supplemented with ampicillin (100μg/ml). Plasmid DNA was prepared from these cultures using a BIOROBOT®9600 workstation (Qiagen, Inc.) and subjected restriction digest checks.The plasmid DNA from one clone having the correct restriction digestpattern was further subjected to sequence analysis and designatedpMBin200.

The I. orientalis YMR226c coding sequence was amplified by PCR using twosynthetic oligonucleotide primers designed to generate an EcoRIrestriction site at the 5′ end and a XmaI restriction site at the 3′ endfor integration into pTrc99A (supra).

Twenty nanograms of pMBin200 plasmid DNA was used as a template in a PCRreaction further containing 1× Phusion HF buffer, fifty picomoles eachof primers 614967 and 614968, 0.2 mM each of dATP, dGTP, dCTP, and dTTP,and 2 units Phusion® Hot Start High-Fidelity DNA Polymerase (Finnzymes,Vantaa, Finland) in a final volume of 50 μl. The amplification reactionwas performed in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific,Inc.) programmed for one cycle at 95° C. for 3 minutes; and 30 cycleseach at 95° C. for 30 seconds, 56.5° C. for 30 seconds, and 72° C. for 1minute. After the 30 cycles, the reaction was incubated at 72° C. for 5minutes and then cooled at 10° C. until further processed.

The 831 bp PCR fragment from the PCR reaction mixture was subjected to1% TBE-agarose gel electrophoresis with ethidium bromide in TBE bufferand the PCR product was cut out of the gel and purified using theNucleoSpin Extract II kit (Macherey-Nagel). The purified fragment wasdigested with EcoRI and XmaI (New England Biolabs) and the plasmidpTrc99A was digested with EcoRI and XmaI, and the resulting fragmentsseparated by 1% TBE-agarose gel electrophoresis followed byvisualization with a DARK READER™ (Clare Chemical Research). The desired4.16 kb fragment of pTrc99A and 819 bp YMR226c fragment was excised fromthe gel with a disposable razor blade and purified using a NucleoSpinExtract II kit (Macherey-Nagel).

Cloning of the DNA fragment containing I. orientalis YMR226c codingsequence into pTrc99A was performed using T4 DNA ligase (New EnglandBiolabs). The reaction mixture contained 1× T4 DNA ligase buffer, 1 μlT4 DNA ligase, 1 μl of the pTrc99A EcoRI/XmaI digested DNA fragmentabove, and 15 μl of the YMR226c EcoRI/XmaI digested PCR product above intotal volume of 20 μl. The reaction mixture was incubated at roomtemperature for 1 hour and subsequently used to transform Solo Pack Goldsupercompetent cells (Agilent Technologies, Inc.) according tomanufacturer's instructions. After a recovery period, two 100 μlaliquots from the transformation mixture were plated onto 150 mm 2XYTplates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C. Recombinant colonies of the transformations wereeach inoculated into 3 ml of LB medium supplemented with ampicillin (100μg/ml).

Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600workstation (Qiagen, Inc.) and analyzed by 1% TBE-agarose gelelectrophoresis following EcoRI/XmaI digestion. The plasmid DNA from oneclone designated pMcTs103 and having the correct restriction digestpattern was further subjected to sequence analysis to confirmintegration of the correct YMR226c coding sequence. From sequencing itwas determined that this YMR226c coding sequence differed from theexpected genomic sequence by 1 base pair.

To correct the 1 base pair mutation in pMcTs103 site directedmutagenesis was performed using pMcTs103 as the template DNA in a PCRreaction using QuikChange II XL Site-Directed Mutagenesis Kit (AgilentTechnologies, Inc.) as described supra using primers 615428 and 614429.

Recombinant colonies of the transformations were each inoculated into 3ml of LB medium supplemented with ampicillin (100 μg/ml). Plasmid DNAwas prepared from these cultures using a BIOROBOT® 9600 workstation(Qiagen, Inc.) and subjected to sequence analysis to confirm the sitedirected mutation in the YMR226c coding sequence. A clone with thecorrect sequence based on sequencing was named pMcTs107.

Example 6 Construction of I. Orientalis YMR226c 3-HPDH Gene Variants

Based on the findings of shown in Example 4, additional 3-HPDH genevariants were constructed using the parent I. orientalis YMR226c homolog(SEQ ID NO: 4).

Site-directed mutagenesis was performed using pMcTs107 (supra) as thetemplate DNA in a PCR reaction using a QuikChange II XL Site-DirectedMutagenesis Kit (Agilent Technologies, Inc.) as described supra usingprimers 615006 and 615007, designed to make amino acid substitutions ofaspartic acid and leucine at positions 45 and 46 of SEQ ID NO: 4,respectively (corresponding to positions 31 and 32 of SEQ ID NO: 2)resulting in the variant mut26 (SEQ ID NO: 80).

Recombinant colonies of the transformations were subjected to sequenceanalysis to confirm the site directed substitutions in the YMR226ccoding sequence. A clone with the correct sequence encoding the variantmut26 (SEQ ID NO: 80) was named pMcTs110.

Site-directed mutagenesis was performed on pMcTs110 in a PCR reactionusing a QuikChange II XL Site-Directed Mutagenesis Kit (AgilentTechnologies, Inc.) as described supra using primers 615004 and 615005,designed to introduce an amino acid substitution of glycine at position20 of SEQ ID NO: 80 (corresponding to position 9 of SEQ ID NO: 2)resulting in the variant mut27 (SEQ ID NO: 81).

Recombinant colonies of the transformations were subjected to sequenceanalysis to confirm the site directed substitutions in the YMR226ccoding sequence and a clone with the correct sequence encoding thevariant mut27 (SEQ ID NO: 81) was named pMcTs112.

Example 7 Cofactor Specificity of Cells Expressing I. Orientalis YMR226c3-HPDH Gene Variants

The I. orientalis gene variants from Example 6 were expressed in MG1655cells and the 3-HPDH cofactor specificity was measured using themalonate semi-aldehyde reductase assay described supra. Results areshown below in Table 5. The mut27 I. orientalis YMR226c 3-HPDH variantexpressed from pMcTs112 (SEQ ID NO: 81) showed increased specificity forNAD(H) over to NADP(H) compared to the parent I. orientalis YMR226c geneproduct expressed from pMcTs107 (SEQ ID NO: 4) and a control lacking aYMR226c gene product (blank expression vector pTrc99A).

TABLE 5 Cloning Serine DeH SA Serine DeH SA Plasmid (uts/mg prot) pH 6(uts/mg prot) pH 8 Name SEQ ID Name NADPH NADH NADPH NADH Control —pTrc99A 53.32 8.46 7.79 6.96 E. coli ydfG (wt) 1 pMeJi9 12137.66 10.25737.62 8.73 mut22 30 pMcTs100 36.08 121.98 7.37 38.01 I. orientalis 4pMcTs107 399.52 8.20 131.39 5.21 YMR226c (wt) mut27 81 pMcTs112 58.02399.06 5.38 18.76

Example 8 Construction of an Expression Vector for Integration of the I.Orientalis YMR226c 3-HPDH Gene at the L. orientalis adh9091 Locus

The I. orientalis YMR226 3-HPDH coding sequence was amplified frompMcTs107 (supra) with primers designed to add flanking 5′ NheI and 3′PacI restriction sites. Fifty nanograms of pMcTs107 plasmid DNA was usedas a template in a PCR reaction further containing 1× Phusion HF buffer,fifty picomoles each of primers 615485 and 615486, 0.2 mM each of dATP,dGTP, dCTP, and dTTP, and 2 units Phusion® Hot Start High-Fidelity DNAPolymerase (Finnzymes) in a final volume of 50 μl. The amplificationreaction was performed in an EPPENDORF® MASTERCYCLER® 5333 (EppendorfScientific, Inc.) programmed for one cycle at 95° C. for 3 minutes; and30 cycles each at 95° C. for 30 seconds, 55° C. for 30 seconds, and 72°C. for 1 minute. After the 30 cycles, the reaction was incubated at 72°C. for 5 minutes and then cooled at 10° C. until further processed.

The 837 bp PCR fragment from PCR reaction mixture was subjected to 1%TBE-agarose gel electrophoresis with ethidium bromide in TBE buffer andthe PCR product was excised from the gel and purified using theNucleoSpin Extract II kit (Macherey-Nagel). The purified fragment wasdigested with NheI and PacI (New England Biolabs) and the plasmidpMBin204 (WO2012/074818) was digested with XbaI and PacI, and theresulting fragments separated by 1% TBE-agarose gel electrophoresisfollowed by visualization with a DARK READER™ (Clare Chemical Research).The desired 8.4 kb fragment of pMBin204 and 827 bp YMR226c fragment wasexcised from the gel and purified using a NucleoSpin Extract II kit(Macherey-Nagel).

Cloning of the DNA fragment containing the coding sequence of the I.orientalis YMR226c 3-HPDH (SEQ ID NO: 4) into pMBin204 was performedusing T4 DNA ligase (New England Biolabs). The reaction mixturecontained 1× T4 DNA ligase buffer, 1 μl T4 DNA ligase, 1 μl of thepMBin204 XbaI and PacI digested DNA fragment above, and 5 μl of theYMR226c NheI and PacI digested PCR product above in total volume of 20μl. The reaction mixture was incubated at room temperature for 1 hourand subsequently used to transform One Shot TOP10 cells (Invitrogen)according to manufacturer's instructions. After a recovery period, two100 μl aliquots from the transformation mixture were plated onto 150 mm2XYT plates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C. Recombinant colonies of the transformations wereeach inoculated into 3 ml of LB medium supplemented with ampicillin (100μg/ml).

Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600workstation (Qiagen, Inc.) and analyzed by restriction digestion. Theplasmid DNA from one clone having the correct restriction digest patternwas further subjected to sequence analysis to confirm the correctYMR226c coding sequence was designated pMcTs108 (FIG. 5).

Example 9 Construction of an Expression Vector for Integration of the I.Orientalis YMR226c 3-HPDH Gene Variants at the L. orientalis adh9091Locus

The coding sequence for the I. orientalis YMR226 variant mut27 (SEQ IDNO: 81) was amplified from pMcTs112 (supra) with primers designed to addflanking 5′ NheI and 3′ PacI restriction sites. Fifty nanograms ofpMcTs112 plasmid DNA was used as a template in a PCR reaction furthercontaining 1× Phusion HF buffer, fifty picomoles each of primers 615485and 615486, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, and 2 unitsPhusion® Hot Start High-Fidelity DNA Polymerase (Finnzymes) in a finalvolume of 50 μl. The amplification reaction was performed in anEPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc.) programmedfor one cycle at 95° C. for 3 minutes; and 30 cycles each at 95° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. After the 30cycles, the reaction was incubated at 72° C. for 5 minutes and thencooled at 10° C. until further processed.

The 837 bp PCR fragment from PCR reaction mixture was subjected to 1%TBE-agarose gel electrophoresis with ethidium bromide in TBE buffer andthe PCR product was cut out of the gel and purified using the NucleoSpinExtract II kit (Macherey-Nagel). The purified fragment was digested withNheI and PacI (New England Biolabs) and the plasmid pMBin204 wasdigested with XbaI and PacI, and the resulting fragments separated by 1%TBE-agarose gel electrophoresis followed by visualization with a DARKREADER™ (Clare Chemical Research). The desired 8.4 kb fragment ofpMBin204 and 827 bp YMR226c variant fragment was excised from the geland purified using a NucleoSpin Extract II kit (Macherey-Nagel).

Cloning of the DNA fragment containing the coding sequence for the I.orientalis YMR226c 3-HPDH variant mut27 into pMBin204 was performedusing T4 DNA ligase (New England Biolabs). The reaction mixturecontained 1× T4 DNA ligase buffer, 1 μl T4 DNA ligase, 1 μl of thepMBin204 XbaI and PacI digested DNA fragment above, and 10 μl of theYMR226c variant NheI and PacI digested PCR product above in total volumeof 20 μl. The reaction mixture was incubated at room temperature for 1hour and subsequently used to transform One Shot TOP10 cells(Invitrogen) according to manufacturer's instructions. After a recoveryperiod, two 100 μl aliquots from the transformation mixture were platedonto 150 mm 2XYT plates supplemented with 100 μg of ampicillin per mland incubated overnight at 37° C. Recombinant colonies of thetransformations were each inoculated into 3 ml of LB medium supplementedwith ampicillin (100 μg/ml).

Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600workstation (Qiagen, Inc.) and analyzed by restriction digestion. Theplasmid DNA from one clone having the correct restriction digest patternwas further subjected to sequence analysis to confirm the correctYMR226c coding sequence and designated pMcTs116 (FIG. 6).

Example 10 Construction of an Expression Vector for Integration of theE. coli ydfG 3-HPDH Gene at the I. Orientalis adh9091 Locus

The coding sequence for the E. coli ydfG 3-HPDH was codon-optimized forI. orientalis DNA, flanked by 5′ XbaI site and 3′ PacI restrictionsites, and provided by GeneArt in a plasmid designated p1045168 (FIG.7). Plasmids p1045168 and pMBin204 (WO2012/074818) were individuallydigested with XbaI and PacI and the resulting fragments separated by 1%TBE-agarose gel electrophoresis and visualized with a DARK READER™(Clare Chemical Research). The desired 8.4 kb fragment of pMBin204 and761 bp E. coli ydfG fragment was excised from the gel and purified usinga NucleoSpin Extract II kit (Macherey-Nagel).

Cloning of the DNA fragment containing the coding sequence of the E.coli ydfG 3-HPDH (SEQ ID NO: 2) into pMBin204 was performed using T4 DNAligase (New England Biolabs). The reaction mixture contained 1× T4 DNAligase buffer, 1 μl T4 DNA ligase, 1 μl of the pMBin204 XbaI and PacIdigested DNA fragment above, and 10 μl of the E. coli ydfG product intotal volume of 20 μl. The reaction mixture was incubated at roomtemperature for 1 hour and subsequently used to transform One Shot TOP10cells (Invitrogen) according to manufacturer's instructions. After arecovery period, two 100 μl aliquots from the transformation mixturewere plated onto 150 mm 2XYT plates supplemented with 100 μg ofampicillin per ml and incubated overnight at 37° C. Recombinant coloniesof the transformations were each inoculated into 3 ml of LB mediumsupplemented with ampicillin (100 μg/ml).

Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600workstation (Qiagen, Inc.) and analyzed by restriction digestion. Theplasmid DNA from one clone having the correct restriction digest patternwas designated pMcTs77 (FIG. 8).

Example 11 Construction of an Expression Vectors for Integration of theE. coli ydfG 3-HPDH Gene Variants at the I. Orientalis adh9091 Locus

The coding sequence for the E. coli ydfG 3-HPDH variant mut6 (SEQ ID NO:12) was codon-optimized for I. orientalis DNA, flanked by 5′ XbaI siteand 3′ PacI restriction sites, and provided by GeneArt in a plasmiddesignated p11AAT5WP (FIG. 9). Fifty nanograms of p11AAT5WP DNA was usedas a template in a PCR reaction further containing 1× Expand buffer,fifty picomoles each of primers 614697 and 614698, 0.2 mM each of dATP,dGTP, dCTP, and dTTP, and 2.6 units Expand High Fidelity Polymerase(Roche) in a final volume of 50 μl. The amplification reaction wasperformed in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific,Inc.) programmed for one cycle at 95° C. for 2 minutes; and 30 cycleseach at 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute. After the 30 cycles, the reaction was incubated at 72° C. for 5minutes and then cooled at 10° C. until further processed.

The 783 bp PCR fragment was digested with XbaI and PacI and cloned intothe 8.4 kb fragment of pMBin204 (supra) also digested with XbaI andPacI. Recombinant clones were screened by restriction digest andsequencing and a clone with the correct sequence was designated pMcTs78(FIG. 10).

Plasmid p1045168 (supra; see also FIG. 7) was subjected to site directedmutagenesis using primers 615892 and 615893 as described supra to changethe coding sequence for the wild-type E. coli ydfG 3-HPDH (SEQ ID NO: 2)into the coding sequence for the E. coli ydfG 3-HPDH variant mut25 (SEQID NO: 33) which contains the substitutions G31 D and R32L. Recombinantcolonies of the transformations were sequenced and a clone encoding the3-HPDH with the correct amino acid changes was named pMcTs111.

Plasmid pMcTs111 was subjected to site directed mutagenesis usingprimers 615890 and 615891 as described supra to change the codingsequence for the E. coli ydfG 3-HPDH variant mut25 (SEQ ID NO: 33) intothe coding sequence for the E. coli ydfG 3-HPDH variant mut22 (SEQ IDNO: 30) which contains the an additional T9G substitution. Recombinantcolonies of the transformations were sequenced and a clone encoding the3-HPDH with the correct amino acid changes was named pMcTs111.Recombinant colonies of the transformations were sequenced and a cloneencoding the 3-HPDH with the correct amino acid changes was namedpMcTs113.

The coding sequence for the E. coli ydfG 3-HPDH variant mut22 (SEQ IDNO: 30) was cloned into pMBin204 by digesting pMcTs113 with XbaI andPacI and ligating the resulting 761 bp fragment of pMcTs113 into theresulting 8.4 kbp fragment of pMBin204 also digested with XbaI and PacIas described supra. Recombinant clones were screened by restrictiondigest and sequencing, and a clone with the correct sequence wasdesignated pMcTs115 (FIG. 11).

Example 12 Construction of an Expression Vectors for Integration of theP. putida mmsB 3-HPDH at the I. Orientalis adh9091 Locus

The coding sequence for the P. putida mmsB 3-HPDH (SEQ ID NO: 82) wascodon-optimized for I. orientalis DNA, flanked by 5′ XbaI site and 3′PacI restriction sites, and provided by GeneArt in a plasmid designatedp11AA2GJP (FIG. 12). Plasmid p11AA2GJP was digested with XbaI and PacIand the 898 bp fragment was cloned into the 8.4 kb fragment of pMBin204also digested with XbaI and PacI as described supra. Several recombinantclones were screened by restriction digest and sequenced. One clone withthe correct sequence was designated pMcTs102 (FIG. 13).

Example 13 Construction of Host Strains Containing an Active 3-HPPathway and Expressing 3-HPDH at the I. Orientalis adh9091 Locus

Approximately 10 μg each of each integration construct pMcTs77, pMcTs78,pMcTs102, pMcTs115 supra was individually digested with ApaI and KpnIand separated by gel electrophoresis on a 1% agarose gel using 89 mMTris base-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer.Approximately 10 μg each of integration constructs pMcTs108 and pMcTs116was digested with ApaI and SacI and separated by gel electrophoresis ona 1% agarose gel using TBE buffer. Fragments of approximately 5348 bpfor pMcTs77, pMcTs78, and pMcTs115; 5485 bp for pMcTs102; and 5408 bpfor pMcTs108 and pMcTs116 were excised and extracted using the QIAquickgel extraction kit (Qiagen, Inc.) according to the manufacturer'sinstructions. The linear constructs from plasmids pMcTs77, pMcTs78,pMcTs102, pMcTs115, pMcTs116, pMcTs108 were transformed into strainMcTs259 (containing an active 3-HP pathway but having a deletion to thenative I. orientalis YMR226c 3-HPDH gene; see WO2012/074818). Severalsingle isolates from each transformation were screened for the site ofintegration as well as confirming that the other loci were stillmodified. The integration at adh9091 was confirmed by PCR using Phire®Plant Direct PCR kit (Finnzymes) according to the manufacturer'sinstructions with primers 614627+612909 and 612908+614626. The PCRproduct using primers 612908+614626 was approximately 1.97 kb forpMcTs77, pMcTs78, pMcTs102, pMcTs115, pMcTs116, and pMcTs108 integrants.The PCR product using primers 614627+612909 was approximately 3.4 kb forpMcTs102 integrants and approximately 3.3 kb for pMcTs77, pMcTs78,pMcTs102, pMcTs115, pMcTs116, pMcTs108 integrants. The integrity of theexisting adh1202 locus and YMR226c locus was verified using primer sets611245+612794 and 611815+612795 for adh1202 locus, and primer set613034+613241 for YMR226c locus. A transformant with the correct sizebands for the PCRs was designed as show below in Table 6.

TABLE 6 Resulting Plasmid 3-HPDH gene host strain pMcTs77 E. coli ydfG(wt) McTs263 pMcTs78 E. coli mut6 McTs265 pMcTs102 P. putida mmsBMcTs276 pMcTs115 E. coli mut22 ShTh100 pMcTs116 I. orientalis mut27ShTh101 pMcTs108 I. orientalis YMR226c (wt) MBin556

Example 14 3-HP Production from Host Strains Containing an Active 3-HPPathway and Expressing 3-HPDH at the I. Orientalis adh9091 Locus

Strains McTs263, McTs265, McTs276, ShTh100, ShTh101, and MBin556 suprawere grown in shake flasks and samples were analyzed for cofactorspecificity (as described supra) and 3-HP production as described inWO2012/074818. Control strains MeJi412 (containing an active 3-HPpathway including the native I. orientalis YMR226c 3-HPDH gene) andMcTs244 (containing an active 3-HP pathway but having a deletion to thenative I. orientalis YMR226c 3-HPDH gene) described in WO2012/074818,were also analyzed for 3-HPDH activity and 3-HP production. The resultsin table 7 show that deletion of the I. orientalis YMR226c gene resultsin no detectable 3-HP production and that 3-HP production can berestored using one copy of a gene encoding a 3-HPDH that has increasedspecificity for NAD(H).

TABLE 7 3HP (g/L)/ 3HPDH SA, pH 8.0 3HPDH SA, pH 6.0 Strain 3-HPDH geneOD600 NADH NADPH NADH NADPH McTs263 E. coli ydfG (wt) 0.08 12.10 18.5647.32 113.91 McTs265 E. coli mut6 0.03 17.95 0.00 90.38 23.15 McTs276 P.putida mmsB 0.06 101.10 0.00 1878.38 193.42 ShTh100 E. coli mut22 0.0617.25 0.00 72.88 24.156 ShTh101 I. orientalis mut27 0.05 18.30 0.00221.67 24.58 MBin556 I. orientalis YMR226c (wt) 0.07 13.78 68.86 70.69292.98 MeJi412 native 0.14 18.86 47.10 96.33 139.91 McTs244 Deletion ofnative 0.00 12.86 0.00 57.45 19.78 YMR226c

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

In some aspects, the invention may be described by the followingnumbered paragraphs:

[1] A 3-HPDH variant, comprising a substitution at one or more (several)positions corresponding to positions 9, 31, 32, 33, 34, 35 and 36 of SEQID NO: 2;

wherein the variant has at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto SEQ ID NO: 2, 4, or 6; and wherein the variant has 3-HPDH activity.

[2] The variant of paragraph [1], which is a variant of a parent 3-HPDHselected from:

a. a polypeptide having at least 60% sequence identity to SEQ ID NO: 2,4, or 6;

b. a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with a polynucleotide having SEQ ID NO: 1, 3, 5,or the full-length complement thereof; and

c. a polypeptide encoded by a polynucleotide having at least 60%sequence identity to SEQ ID NO: 1, 3, or 5.

[3] The variant of paragraph [2], wherein the parent 3-HPDH has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, 4, or6.[4] The variant of paragraph [2] or [3], wherein the parent 3-HPDH isencoded by a polynucleotide that hybridizes under at least lowstringency conditions, e.g., medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with a polynucleotide having SEQ ID NO: 1, 3, 5,or the full-length complement thereof.[5] The variant of any of paragraphs [2]-[4], wherein the parent 3-HPDHis encoded by a polynucleotide having at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 1, 3, or 5[6] The variant of any of paragraphs 2-5, wherein the parent 3-HPDHcomprises or consists of SEQ ID NO: 2, 4, or 6.[7] The variant of any of paragraphs [2]-[6], which has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% identity, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof the parent 3-HPDH.[8] The variant of any of paragraphs [1]-[7], wherein the number ofsubstitutions is 1-20, e.g., 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 substitutions.[9] The variant of any of paragraphs [1]-[8], comprising a substitutionat a position corresponding to position 9 of SEQ ID NO: 2.[10] The variant of any one of paragraphs [1]-[9], comprising a Gly at aposition corresponding to position 9 of SEQ ID NO: 2.[11] The variant of any of paragraphs [1]-[10], comprising asubstitution at a position corresponding to position 31 of SEQ ID NO: 2.[12] The variant of any one of paragraphs [1]-[11], comprising an Asp orGlu at a position corresponding to position 31 of SEQ ID NO: 2.[13] The variant of any of paragraphs [1]-[12], comprising asubstitution at a position corresponding to position 32 of SEQ ID NO: 2.[14] The variant of any one of paragraphs [1]-[13], comprising a Leu ata position corresponding to position 32 of SEQ ID NO: 2.[15] The variant of any of paragraphs [1]-[14], comprising asubstitution at a position corresponding to position 33 of SEQ ID NO: 2.[16] The variant of any one of paragraphs [1]-[15], comprising a Ser orAsn at a position corresponding to position 33 of SEQ ID NO: 2.[17] The variant of any of paragraphs [1]-[16], comprising asubstitution at a position corresponding to position 34 of SEQ ID NO: 2.[18] The variant of any one of paragraphs [1]-[17], comprising an Ala orPro at a position corresponding to position 34 of SEQ ID NO: 2.[19] The variant of any of paragraphs [1]-[18], comprising asubstitution at a position corresponding to position 35 of SEQ ID NO: 2.[20] The variant of any one of paragraphs [1]-[19], comprising an Ala orAsp at a position corresponding to position 35 of SEQ ID NO: 2.[21] The variant of any of paragraphs [1]-[20], comprising asubstitution at a position corresponding to position 36 of SEQ ID NO: 2.[22] The variant of any one of paragraphs [1]-[21], comprising an Ala ata position corresponding to position 36 of SEQ ID NO: 2.[23] The variant of any of paragraphs [1]-[22], comprising at least twosubstitutions at positions corresponding to any of positions 9, 31, 32,33, 34, 35, and 36 of SEQ ID NO: 2.[24] The variant of any of paragraphs [1]-[22], comprising at leastthree substitutions at positions corresponding to any of positions 9,31, 32, 33, 34, 35, and 36 of SEQ ID NO: 2.[25] The variant of any of paragraphs [1]-[22], comprising at least foursubstitutions at positions corresponding to any of positions 9, 31, 32,33, 34, 35, and 36 of SEQ ID NO: 2.[26] The variant of any of paragraphs [1]-[22], comprising at least fivesubstitutions at positions corresponding to any of positions 9, 31, 32,33, 34, 35, and 36 of SEQ ID NO: 2.[27] The variant of any of paragraphs [1]-[22], comprising at least sixsubstitutions at positions corresponding to any of positions 9, 31, 32,33, 34, 35, and 36 of SEQ ID NO: 2.[28] The variant of any of paragraphs [1]-[22], comprising sevensubstitutions at positions corresponding to positions 9, 31, 32, 33, 34,35, and 36 of SEQ ID NO: 2.[29] The variant of any of paragraphs [1]-[24], comprising one or moresubstitutions selected from T/S9G, G/A31D/E, R32L, R33S/N, L/K/Q34A/P,E35D/A, and K/R36A corresponding to positions of SEQ ID NO: 2.[30] The variant of any of paragraphs [1]-[29], further comprising adeletion at a position corresponding to position 10 of SEQ ID NO: 2.[31] The variant of any one of paragraphs [1]-[30], wherein the variantcomprises or consists of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 80, or 81.[32] The variant of any of paragraphs [1]-[31], wherein the variant hasincreased specificity for NAD(H) compared to NADP(H) (e.g., greater than2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,or 1000-fold specificity for NAD(H) compared to NADP(H)).[33] The variant of any one of paragraphs [1]-[32], wherein the variantis isolated.[34] A polynucleotide (e.g., an isolated polynucleotide) encoding thevariant of any of paragraphs [1]-[32].[35] A nucleic acid construct comprising the polynucleotide of paragraph[34].[36] An expression vector comprising the polynucleotide of paragraph[34].[37] A host cell comprising the polynucleotide of paragraph [35].[38] A method of producing a 3-HPDH variant, comprising:

a. cultivating the host cell of paragraph [37] under conditions suitablefor expression of the variant; and

b. recovering the variant.

[39] A transgenic plant, plant part or plant cell transformed with thepolynucleotide of paragraph [34].

[40] A method of producing a variant of any of paragraphs [1]-[34],comprising:

a. cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the variant under conditions conducive forproduction of the variant; and

b. recovering the variant.

[41] A method for obtaining the 3-HPDH variant of any one of paragraphs[1]-[34], comprising introducing into a parent 3-HPDH a substitution atone or more positions corresponding to positions 9, 31, 32, 33, 34, 35,and 36 of SEQ ID NO: 2; and recovering the variant.[42] A polypeptide having 3-HPDH activity, wherein the polypeptide is:

a. a polypeptide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, sequenceidentity to SEQ ID NO: 2, 4, or 6;

b. a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, e.g., medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with a polynucleotide having SEQ ID NO: 1, 3, 5,or the full-length complement thereof; or

c. a polypeptide encoded by a polynucleotide having at least 60%sequence identity to SEQ ID NO: 1, 3, or 5;

and wherein the polypeptide has increased specificity for NAD(H)compared to NADP(H) (e.g., greater than 2-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold specificityfor NAD(H) compared to NADP(H)).

[43] The polypeptide of paragraph [42], having at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to SEQ ID NO: 2.[44] The polypeptide of paragraph [42] or [43], which is encoded by apolynucleotide that hybridizes under at least low stringency conditions,e.g., medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with apolynucleotide having SEQ ID NO: 1 or the full-length complementthereof.[45] The polypeptide of any of paragraphs [42]-[44], which is encoded bya polynucleotide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 1.[46] The polypeptide of any of paragraphs [42]-[45], wherein at leastone of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differs from SEQ ID NO: 2.[47] The polypeptide of any of paragraphs [42]-[45], wherein at leasttwo of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 2.[48] The polypeptide of any of paragraphs [42]-[45], wherein at leastthree of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 2.[49] The polypeptide of any of paragraphs [42]-[45], wherein at leastfour of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 2.[50] The polypeptide of any of paragraphs [42]-[45], wherein at leastfive of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 2.[51] The polypeptide of any of paragraphs [42]-[45], wherein at leastsix of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 2.[52] The polypeptide of any of paragraphs [42]-[45], wherein all ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differ from SEQ ID NO: 2.[53] The polypeptide of any of paragraphs [42]-[52], having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4.[54] The polypeptide of any of paragraphs [42]-[53], which is encoded bya polynucleotide that hybridizes under at least low stringencyconditions, e.g., medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with a polynucleotide having SEQ ID NO: 3 or the full-lengthcomplement thereof.[55] The polypeptide of any of paragraphs [42]-[54], which is encoded bya polynucleotide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 3.[56] The polypeptide of any of paragraphs [42]-[56], wherein at leastone of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differs from SEQ ID NO: 4.[57] The polypeptide of any of paragraphs [42]-[55], wherein at leasttwo of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 4.[58] The polypeptide of any of paragraphs [42]-[55], wherein at leastthree of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 4.[59] The polypeptide of any of paragraphs [42]-[55], wherein at leastfour of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 4.[60] The polypeptide of any of paragraphs [42]-[55], wherein at leastfive of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 4.[61] The polypeptide of any of paragraphs [42]-[55], wherein at leastsix of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 4.[62] The polypeptide of any of paragraphs [42]-[55], wherein all ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differ from SEQ ID NO: 4.[63] The polypeptide of any of paragraphs [42]-[62], having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6.[64] The polypeptide of any of paragraphs [42]-[63], which is encoded bya polynucleotide that hybridizes under at least low stringencyconditions, e.g., medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with a polynucleotide having SEQ ID NO: 5 or the full-lengthcomplement thereof.[65] The polypeptide of any of paragraphs [42]-[64], which is encoded bya polynucleotide having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 5.[66] The polypeptide of any of paragraphs [42]-[65], wherein at leastone of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differs from SEQ ID NO: 6.[67] The polypeptide of any of paragraphs [42]-[65], wherein at leasttwo of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 6.[68] The polypeptide of any of paragraphs [42]-[65], wherein at leastthree of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 6.[69] The polypeptide of any of paragraphs [42]-[65], wherein at leastfour of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 6.[70] The polypeptide of any of paragraphs [42]-[65], wherein at leastfive of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 6.[71] The polypeptide of any of paragraphs [42]-[65], wherein at leastsix of positions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ IDNO: 2 differ from SEQ ID NO: 6.[72] The polypeptide of any of paragraphs [42]-[65], wherein all ofpositions 9, 31, 32, 33, 34, 35 and 36 corresponding to SEQ ID NO: 2differ from SEQ ID NO: 6.[73] The polypeptide of any one of paragraphs [42]-[72], comprising aGly at a position corresponding to position 9 of SEQ ID NO: 2.[74] The polypeptide of any one of paragraphs [42]-[73], comprising anAsp or Glu at a position corresponding to position 31 of SEQ ID NO: 2.[75] The polypeptide of any one of paragraphs [42]-[74], comprising aLeu at a position corresponding to position 32 of SEQ ID NO: 2.[76] The polypeptide of any one of paragraphs [42]-[75], comprising aSer or Asn at a position corresponding to position 33 of SEQ ID NO: 2.[77] The polypeptide of any one of paragraphs [42]-[76], comprising anAla or Pro at a position corresponding to position 34 of SEQ ID NO: 2.[78] The polypeptide of any one of paragraphs [42]-[77], comprising anAla or Asp at a position corresponding to position 35 of SEQ ID NO: 2.[79] The polypeptide of any one of paragraphs [42]-[78], comprising anAla at a position corresponding to position 36 of SEQ ID NO: 2.[80] The polypeptide of any one of paragraphs [42]-[79], furthercomprising a deletion at a position corresponding to position 10 of SEQID NO: 2.[81] The polypeptide of any one of paragraphs [42]-[80], wherein thepolypeptide comprises or consists of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,80, or 81.[82] The polypeptide of any one of paragraphs [42]-[82], wherein thepolypeptide is isolated.[83] A polynucleotide (e.g., an isolated polynucleotide) encoding thepolypeptide of any of paragraphs [42]-[82].[84] A nucleic acid construct comprising the polynucleotide of paragraph[83].[85] An expression vector comprising the polynucleotide of paragraph[83].[86] A host cell comprising the polynucleotide of paragraph [83].[87] A method of producing the polypeptide of any of paragraphs[42]-[82], comprising:

a. cultivating a host cell comprising a polynucleotide encoding thepolypeptide under conditions suitable for expression of the polypeptide;and

b. recovering the polypeptide.

[88] A transgenic plant, plant part or plant cell transformed with thepolynucleotide of paragraph [83].

[89] A method of producing the polypeptide of any of paragraphs[42]-[82], comprising:

a. cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and

b. recovering the variant.

[90] A method for obtaining the polypeptide of any of paragraphs[42]-[82], comprising introducing into a parent 3-HPDH a substitution atone or more positions corresponding to positions 9, 31, 32, 33, 34, 35,and 36 of SEQ ID NO: 2; and recovering the polypeptide.[91] The host cell of paragraph [37] or [86], wherein cell isprokaryotic.[92] The host cell of paragraph [37] or [86], wherein the cell iseukaryotic.[93] The host cell of paragraph [92], wherein the cell is a yeast cell.[94] The host cell of paragraph [93], wherein the cell belongs to agenus selected from Issatchenkia, Candida, Kluyveromyces, Pichia,Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and Saccharomyces.[95] The host cell of paragraph [94], wherein the cell is selected fromI. orientalis, C. lambica, and S. bulderi.[96] The host cell of any of paragraphs [37], [86], or [91]-[95],wherein the cell comprises an active 3-HP pathway.[97] The host cell of any of paragraphs [37], [86], or [91]-[96],wherein the cell comprises:

PEP carboxylase activity or pyruvate carboxylase activity;

aspartate aminotransferase activity;

aspartate decarboxylase activity; and

beta-alanine/alpha-ketoglutarate aminotransferase (BAAT) acitivity.

[98] The host cell of paragraph [37], [86], or [91]-[97], wherein thecell comprises one or more heterologous polynucleotides selected from:

a heterologous polynucleotide that encodes a PEP carboxylase,

a heterologous polynucleotide that encodes a pyruvate carboxylase,

a heterologous polynucleotide that encodes a aspartate aminotransferase,

a heterologous polynucleotide that encodes a aspartate decarboxylase,and

a heterologous polynucleotide that encodes a BAAT.

[99] A method of producing 3-HP, comprising:

a. cultivating the host cell of any of paragraphs [37], [86], and[91]-[98] under conditions conducive for production of 3-HP; and

b. recovering the 3-HP.

[100] A host cell comprising an active 3-HP pathway and a heterologouspolynucleotide encoding a 3-HPDH having at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to SEQ ID NO: 82.[101] The host cell of paragraph [100], wherein cell is prokaryotic.[102] The host cell of paragraph [100], wherein the cell is eukaryotic.[103] The host cell of paragraph [102], wherein the cell is a yeastcell.[104] The host cell of paragraph [103], wherein the cell belongs to agenus selected from Issatchenkia, Candida, Kluyveromyces, Pichia,Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and Saccharomyces.[105] The host cell of paragraph [104], wherein the cell is selectedfrom I. orientalis, C. lambica, and S. bulderi.[106] The host cell of any of paragraphs [100]-[105], wherein the cellcomprises:

PEP carboxylase activity or pyruvate carboxylase activity;

aspartate aminotransferase activity;

aspartate decarboxylase activity; and

beta-alanine/alpha-ketoglutarate aminotransferase (BAAT) activity.

[107] The host cell of paragraph [100]-[106], wherein the cell comprisesone or more heterologous polynucleotides selected from:

a heterologous polynucleotide that encodes a PEP carboxylase,

a heterologous polynucleotide that encodes a pyruvate carboxylase,

a heterologous polynucleotide that encodes a aspartate aminotransferase,

a heterologous polynucleotide that encodes a aspartate decarboxylase,and

a heterologous polynucleotide that encodes a BAAT.

[108] A method of producing 3-HP, comprising:

a. cultivating the host cell of any of paragraphs [100]-[107] underconditions conducive for production of 3-HP; and

b. recovering the 3-HP.

What is claimed is:
 1. A 3-HPDH variant, comprising a substitution atone or more positions corresponding to positions 9, 31, 32, 33, 34, 35and 36 of SEQ ID NO: 2; wherein the variant has at least 80% sequenceidentity to SEQ ID NO: 2, 4, or 6; and wherein the variant has 3-HPDHactivity.
 2. The variant of claim 1, comprising a substitution at aposition corresponding to position 9 of SEQ ID NO:
 2. 3. The variant ofclaim 1, comprising a Gly at a position corresponding to position 9 ofSEQ ID NO:
 2. 4. The variant of claim 1, comprising a substitution at aposition corresponding to position 31 of SEQ ID NO:
 2. 5. The variant ofclaim 1, comprising an Asp or Glu at a position corresponding toposition 31 of SEQ ID NO:
 2. 6. The variant of claim 1, comprising asubstitution at a position corresponding to position 32 of SEQ ID NO: 2.7. The variant of claim 1, comprising a Leu at a position correspondingto position 32 of SEQ ID NO:
 2. 8. The variant of claim 1, wherein thevariant comprises or consists of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 80,or
 81. 9. The variant of claim 1, wherein the variant has increasedspecificity for NAD(H) compared to NADP(H).
 10. A polypeptide having3-HPDH activity, wherein the polypeptide has sequence identity to SEQ IDNO: 2, 4, or 6; and wherein the polypeptide has increased specificityfor NAD(H) compared to NADP(H).
 11. The polypeptide of claim 10, whereinthe polypeptide has at least 90% sequence identity to SEQ ID NO: 2, 4,or
 6. 12. The polypeptide of claim 10, wherein the polypeptide has atleast 95% sequence identity to SEQ ID NO: 2, 4, or
 6. 13. Thepolypeptide of claim 10, wherein the polypeptide has at least 98%sequence identity to SEQ ID NO: 2, 4, or
 6. 14. The polypeptide of claim10, wherein the polypeptide has greater than 10-fold specificity forNAD(H) compared to NADP(H).
 15. The 3-HPDH variant of claim 1, whereinthe variant has at least 90% sequence identity to SEQ ID NO: 2, 4, or 6.16. The 3-HPDH variant of claim 1, wherein the variant has at least 95%sequence identity to SEQ ID NO: 2, 4, or
 6. 17. The 3-HPDH variant ofclaim 1, wherein the variant has at least 98% sequence identity to SEQID NO: 2, 4, or
 6. 18. The 3-HPDH variant of claim 9, wherein thevariant has greater than 10-fold specificity for NAD(H) compared toNADP(H).
 19. The 3-HPDH variant of claim 1, wherein the variantcomprises substitutions at positions corresponding to positions 9, 31,and 32 of SEQ ID NO:
 2. 20. The 3-HPDH variant of claim 1, wherein thevariant comprising a Gly at a position corresponding to position 9 ofSEQ ID NO: 2; an Asp or Glu at a position corresponding to position 31of SEQ ID NO: 2; and a Leu at a position corresponding to position 32 ofSEQ ID NO: 2.